Halley's+Notebook

=6/2/2014=
 * Reverse Transcription (see 1.17.2014 for detailed method) of 2012 & 2013, July & Sept Field samples**


 * Count || Sample || ng/ul || ug/ul || V (ul) of RNA = 1ug || DEPC 0.1% (ul) || Total Rxn ||
 * 1 || 2013_61 || 120.31 || 0.12031 || 8.31 || 9.4 || 17.75 ||
 * 2 || 2013_62 || 178.53 || 0.17853 || 5.60 || 12.1 || 17.75 ||
 * 3 || 2013_63 || 164.64 || 0.16464 || 6.07 || 11.7 || 17.75 ||
 * 4 || 2013_64 || 169.92 || 0.16992 || 5.89 || 11.9 || 17.75 ||
 * 5 || 2013_65 || 117.92 || 0.11792 || 8.48 || 9.3 || 17.75 ||
 * 6 || 2013_66 || 158.87 || 0.15887 || 6.29 || 11.5 || 17.75 ||
 * 7 || 2013_67 || 127.7 || 0.1277 || 7.83 || 9.9 || 17.75 ||
 * 8 || 2013_68 || 165.15 || 0.16515 || 6.06 || 11.7 || 17.75 ||
 * 9 || 2013_69 || 77.51 || 0.07751 || 12.90 || 4.8 || 17.75 ||
 * 10 || 2013_70 || 70.51 || 0.07051 || 14.18 || 3.6 || 17.75 ||
 * 11 || 2013_71 || 76.28 || 0.07628 || 13.11 || 4.6 || 17.75 ||
 * 12 || 2013_72 || 179.23 || 0.17923 || 5.58 || 12.2 || 17.75 ||
 * 13 || 2013_73 || 169.95 || 0.16995 || 5.88 || 11.9 || 17.75 ||
 * 14 || 2013_74 || 135.52 || 0.13552 || 7.38 || 10.4 || 17.75 ||
 * 15 || 2013_75 || 93.44 || 0.09344 || 10.70 || 7.0 || 17.75 ||
 * 16 || 2013_21 || 155.64 || 0.15564 || 6.43 || 11.3 || 17.75 ||
 * 17 || 2013_22 || 159.02 || 0.15902 || 6.29 || 11.5 || 17.75 ||
 * 18 || 2013_24 || 168.19 || 0.16819 || 5.95 || 11.8 || 17.75 ||
 * 19 || 2013_25 || 172.36 || 0.17236 || 5.80 || 11.9 || 17.75 ||
 * 20 || 2013_26 || 148.32 || 0.14832 || 6.74 || 11.0 || 17.75 ||
 * 21 || 2013_27 || 148.01 || 0.14801 || 6.76 || 11.0 || 17.75 ||
 * 22 || 2013_28 || 95.41 || 0.09541 || 10.48 || 7.3 || 17.75 ||
 * 23 || 2013_29 || 136.3 || 0.1363 || 7.34 || 10.4 || 17.75 ||
 * 24 || 2013_30 || 182.7 || 0.1827 || 5.47 || 12.3 || 17.75 ||
 * 25 || 2012_21 || 143.88 || 0.14388 || 6.95 || 10.8 || 17.75 ||
 * 26 || 2012_22 || 153.18 || 0.15318 || 6.53 || 11.2 || 17.75 ||
 * 27 || 2012_23 || 154.13 || 0.15413 || 6.49 || 11.3 || 17.75 ||
 * 28 || 2012_24 || 159.87 || 0.15987 || 6.26 || 11.5 || 17.75 ||
 * 29 || 2012_25 || 159.46 || 0.15946 || 6.27 || 11.5 || 17.75 ||
 * 30 || 2012_26 || 175.49 || 0.17549 || 5.70 || 12.1 || 17.75 ||
 * 31 || 2012_27 || 63.83 || 0.06383 || 15.67 || 2.1 || 17.75 ||
 * 32 || 2012_28 || 143.29 || 0.14329 || 6.98 || 10.8 || 17.75 ||
 * 33 || 2012_29 || 133.67 || 0.13367 || 7.48 || 10.3 || 17.75 ||
 * 34 || 2012_30 || 87.77 || 0.08777 || 11.39 || 6.4 || 17.75 ||
 * 35 || 2012_31 || 172.68 || 0.17268 || 5.79 || 12.0 || 17.75 ||
 * 36 || 2012_32 || 152.56 || 0.15256 || 6.55 || 11.2 || 17.75 ||
 * 37 || 2012_33 || 155.19 || 0.15519 || 6.44 || 11.3 || 17.75 ||
 * 38 || 2012_34 || 107.45 || 0.10745 || 9.31 || 8.4 || 17.75 ||
 * 39 || 2012_35 || 151.32 || 0.15132 || 6.61 || 11.1 || 17.75 ||
 * 40 || 2012_61 || 165.44 || 0.16544 || 6.04 || 11.7 || 17.75 ||
 * 41 || 2012_62 || 130.12 || 0.13012 || 7.69 || 10.1 || 17.75 ||
 * 42 || 2012_63 || 171.02 || 0.17102 || 5.85 || 11.9 || 17.75 ||
 * 43 || 2012_64 || 74.99 || 0.07499 || 13.34 || 4.4 || 17.75 ||
 * 44 || 2012_65 || 163.03 || 0.16303 || 6.13 || 11.6 || 17.75 ||
 * 45 || 2012_66 || 159.56 || 0.15956 || 6.27 || 11.5 || 17.75 ||
 * 46 || 2012_67 || 137.52 || 0.13752 || 7.27 || 10.5 || 17.75 ||
 * 47 || 2012_68 || 169.18 || 0.16918 || 5.91 || 11.8 || 17.75 ||
 * 48 || 2012_69 || 118.47 || 0.11847 || 8.44 || 9.3 || 17.75 ||
 * 49 || 2012_70 || 168.58 || 0.16858 || 5.93 || 11.8 || 17.75 ||
 * 50 || 2012_71 || 92.8 || 0.0928 || 10.78 || 7.0 || 17.75 ||
 * 51 || 2012_72 || 170.31 || 0.17031 || 5.87 || 11.9 || 17.75 ||
 * 52 || 2012_73 || 158.76 || 0.15876 || 6.30 || 11.5 || 17.75 ||
 * 53 || 2012_74 || 200.8 || 0.2008 || 4.98 || 12.8 || 17.75 ||
 * 54 || 2012_75 || 146.04 || 0.14604 || 6.85 || 10.9 || 17.75 ||
 * Mean cDNA results = 1196.5 ng/ul** (260/280 = 1.72; 260/230 = 1.66)

= = =5/29/2014=
 * qPCR on purified RNA (20ul rnx)** **2012 & 2013, July & Sept Field samples**

54 samples 1 NTC 1 gDNA 2 error = 59 total rxns

gDNA template = 0.5ul pureRNA template = 0.5ul

__ //Master Mix// //EF-1a P5// ____ //:// __ 2x Sso Fast Evogreen 10 x 59 = 590ul Forward primer 2-1 0.5 x 59 = 29.5ul Reverse primer 2-1 0.5 x 59 = 29.5ul H20 DEPC 0.1% 8.5 x 59 = 501.5ul

//Results:// Clean RNA

=5/28/2014= Nanospec results: =4/16-4/17/2014=
 * Standard DNA-free protocol (see 1.16.2014 for details) on 2012 & 2013 July and September Field Samples**
 * Sample ID || ng/ul || 260/280 || 260/230 ||
 * 2013_61 || 120.31 || 1.92 || 0.94 ||
 * 2013_62 || 178.53 || 1.95 || 1.58 ||
 * 2013_63 || 164.64 || 1.94 || 1.22 ||
 * 2013_64 || 169.92 || 1.95 || 0.84 ||
 * 2013_65 || 117.92 || 1.92 || 1.04 ||
 * 2013_66 || 158.87 || 1.92 || 1.31 ||
 * 2013_67 || 127.7 || 1.92 || 1.39 ||
 * 2013_68 || 165.15 || 1.92 || 1.32 ||
 * 2013_69 || 77.51 || 1.85 || 0.83 ||
 * 2013_70 || 70.51 || 1.85 || 1.14 ||
 * 2013_71 || 76.28 || 1.86 || 1.09 ||
 * 2013_72 || 179.23 || 1.92 || 0.92 ||
 * 2013_73 || 169.95 || 1.95 || 1.67 ||
 * 2013_74 || 135.52 || 1.92 || 1.24 ||
 * 2013_75 || 93.44 || 1.89 || 1.00 ||
 * 2013_21 || 155.64 || 1.94 || 1.42 ||
 * 2013_22 || 159.02 || 1.89 || 0.99 ||
 * 2013_24 || 168.19 || 1.94 || 1.31 ||
 * 2013_25 || 172.36 || 1.94 || 1.26 ||
 * 2013_26 || 148.32 || 1.96 || 1.53 ||
 * 2013_27 || 148.01 || 1.93 || 1.52 ||
 * 2013_28 || 95.41 || 1.94 || 1.35 ||
 * 2013_29 || 136.3 || 2.00 || 1.64 ||
 * 2013_30 || 182.7 || 2.00 || 1.36 ||
 * 2012_21 || 143.88 || 1.92 || 1.45 ||
 * 2012_22 || 153.18 || 1.92 || 1.38 ||
 * 2012_23 || 154.13 || 1.93 || 1.23 ||
 * 2012_24 || 159.87 || 1.91 || 1.32 ||
 * 2012_25 || 159.46 || 1.92 || 1.41 ||
 * 2012_26 || 175.49 || 1.94 || 1.36 ||
 * 2012_27 || 63.83 || 1.79 || 0.53 ||
 * 2012_28 || 143.29 || 1.95 || 1.48 ||
 * 2012_29 || 133.67 || 1.94 || 1.53 ||
 * 2012_30 || 87.77 || 1.86 || 1.02 ||
 * 2012_31 || 172.68 || 1.91 || 0.92 ||
 * 2012_32 || 152.56 || 1.91 || 1.32 ||
 * 2012_33 || 155.19 || 1.93 || 1.28 ||
 * 2012_34 || 107.45 || 1.94 || 1.43 ||
 * 2012_35 || 151.32 || 1.93 || 1.29 ||
 * 2012_61 || 165.44 || 1.93 || 1.41 ||
 * 2012_62 || 130.12 || 1.9 || 1.32 ||
 * 2012_63 || 171.02 || 1.94 || 1.51 ||
 * 2012_64 || 74.99 || 1.86 || 0.99 ||
 * 2012_65 || 163.03 || 1.89 || 1.3 ||
 * 2012_66 || 159.56 || 1.91 || 1.34 ||
 * 2012_67 || 137.52 || 1.93 || 1.31 ||
 * 2012_68 || 169.18 || 1.93 || 1.52 ||
 * 2012_69 || 118.47 || 1.91 || 1.13 ||
 * 2012_70 || 168.58 || 1.93 || 1.39 ||
 * 2012_71 || 92.8 || 1.9 || 1.24 ||
 * 2012_72 || 170.31 || 1.94 || 1.48 ||
 * 2012_73 || 158.76 || 1.9 || 1.09 ||
 * 2012_74 || 200.8 || 1.94 || 1.52 ||
 * 2012_75 || 146.04 || 1.92 || 1.21 ||

RNA Extraction 2012 & 2013 Field samples (July & September) (//see 1.15.2014 for detailed methods)//
Nanodrop:
 * Sample || ng/ul || 260/280 || 260/230 ||
 * 2013_61 || 168.79 || 1.84 || 1.36 ||
 * 2013_62 || 446.01 || 1.85 || 1.98 ||
 * 2013_63 || 402.04 || 1.91 || 1.57 ||
 * 2013_64 || 254.12 || 1.92 || 0.84 ||
 * 2013_65 || 167.86 || 1.81 || 1.69 ||
 * 2013_66 || 237.47 || 1.84 || 1.83 ||
 * 2013_67 || 178.7 || 1.8 || 1.85 ||
 * 2013_68 || 313.62 || 1.89 || 1.67 ||
 * 2013_69 || 109.11 || 1.73 || 1.55 ||
 * 2013_70 || 99.51 || 1.72 || 1.73 ||
 * 2013_71 || 106.83 || 1.71 || 1.74 ||
 * 2013_72 || 247.48 || 1.9 || 1.07 ||
 * 2013_73 || 1944.77 || 1.97 || 2.13 ||
 * 2013_74 || 312.59 || 1.88 || 1.85 ||
 * 2013_75 || 231.14 || 1.86 || 1.68 ||
 * 2013_21 || 366.7 || 1.86 || 1.87 ||
 * 2013_22 || 287.7 || 1.9 || 1.24 ||
 * 2013_24 || 315.46 || 1.93 || 1.13 ||
 * 2013_25 || 408.68 || 1.89 || 1.75 ||
 * 2013_26 || 756.4 || 1.97 || 2.03 ||
 * 2013_27 || 625.07 || 1.95 || 2.04 ||
 * 2013_28 || 827.09 || 1.97 || 2.02 ||
 * 2013_29 || 1359.92 || 2 || 2.19 ||
 * 2013_30 || 456.71 || 1.86 || 1.67 ||
 * 2012_21 || 444.88 || 1.85 || 1.95 ||
 * 2012_22 || 251.48 || 1.86 || 1.79 ||
 * 2012_23 || 293.23 || 1.9 || 1.11 ||
 * 2012_24 || 259.93 || 1.87 || 1.69 ||
 * 2012_25 || 298.81 || 1.87 || 1.82 ||
 * 2012_26 || 339.59 || 1.89 || 1.66 ||
 * 2012_27 || 113.63 || 1.77 || 0.79 ||
 * 2012_28 || 552 || 1.94 || 1.91 ||
 * 2012_29 || 563.57 || 1.95 || 2.06 ||
 * 2012_30 || 181.11 || 1.84 || 1.77 ||
 * 2012_31 || 245.64 || 1.93 || 0.57 ||
 * 2012_32 || 588.03 || 1.98 || 1.32 ||
 * 2012_33 || 204.57 || 1.9 || 0.78 ||
 * 2012_34 || 1329.69 || 2.01 || 2.14 ||
 * 2012_35 || 409.82 || 1.9 || 1.59 ||
 * 2012_61 || 311.98 || 1.86 || 1.9 ||
 * 2012_62 || 162.11 || 1.78 || 1.79 ||
 * 2012_63 || 289.12 || 1.89 || 1.53 ||
 * 2012_64 || 103.39 || 1.73 || 1.15 ||
 * 2012_65 || 263.36 || 1.86 || 1.8 ||
 * 2012_66 || 276.73 || 1.87 || 1.7 ||
 * 2012_67 || 181.5 || 1.82 || 1.74 ||
 * 2012_68 || 366.91 || 1.87 || 2.05 ||
 * 2012_69 || 158.96 || 1.78 || 1.5 ||
 * 2012_70 || 387.15 || 1.89 || 1.81 ||
 * 2012_71 || 132.72 || 1.75 || 1.8 ||
 * 2012_72 || 388.71 || 1.9 || 1.69 ||
 * 2012_73 || 312.09 || 1.9 || 1.54 ||
 * 2012_74 || 278.13 || 1.87 || 1.84 ||
 * 2012_74 || 203.24 || 1.83 || 1.55 ||
 * 2012_75 || 199.53 || 1.84 || 1.42 ||

=3/29/2014=

qPCR - cDNA 2013 Field samples 98-99 & 2013 Field samples 99-100 (20ul rxn) HIF-1a & EF-1a

5 samples 5 duplicates 2 NTC 1 error = 13 total rxns

cDNA template = 1ul

__ //Master Mix// //HIF-1a P2-1// ____ //:// __ 2x Sso Fast Evogreen 10 x 13 = 130ul Forward primer 2-1 0.5 x 13 = 6.5ul Reverse primer 2-1 0.5 x 13 = 6.5ul H20 DEPC 0.1% 8 x 13 = 104ul

__ //Master Mix EF////-1a P2// ____ //:// __ 2x Sso Fast Evogreen 10 x 13 = 130ul Forward primer 2 0.5 x 13 = 6.5ul Reverse primer 2 0.5 x 13 = 6.5ul H20 DEPC 0.1% 8 x 13 = 104ul

qPCR - cDNA 2013 Field samples 1-43 & 44-97 (20ul rxn) HIF-1a & EF-1a

23 samples 23 duplicates 2 NTC 2 error = 50 total rxns

cDNA template = 1ul

__//Master Mix// //HIF-1a P2-1//____//://__ 2x Sso Fast Evogreen 10 x 50 = 500ul Forward primer 2-1 0.5 x 50 = 25ul Reverse primer 2-1 0.5 x 50 = 25ul H20 DEPC 0.1% 8 x 50 = 400ul

__//Master Mix EF////-1a P2//____//://__ 2x Sso Fast Evogreen 10 x 50 = 500ul Forward primer 2 0.5 x 50 = 25ul Reverse primer 2 0.5 x 50 = 25ul H20 DEPC 0.1% 8 x 50 = 400ul

=3/31/2014= qPCR - cDNA 2012 Field samples 1-49 & 50-98(20ul rxn) HIF-1a & EF-1a

23 samples 23 duplicates 2 NTC 2 error = 50 total rxns

cDNA template = 1ul

__//Master Mix// //HIF-1a P2-1//____//://__ 2x Sso Fast Evogreen 10 x 50 = 500ul Forward primer 2-1 0.5 x 50 = 25ul Reverse primer 2-1 0.5 x 50 = 25ul H20 DEPC 0.1% 8 x 50 = 400ul

__//Master Mix EF////-1a P2//____//://__ 2x Sso Fast Evogreen 10 x 50 = 500ul Forward primer 2 0.5 x 50 = 25ul Reverse primer 2 0.5 x 50 = 25ul H20 DEPC 0.1% 8 x 50 = 400ul = = =3/29/2014=

Reverse Transcription
2013 Field samples

=3/28/2014=

Reverse Transcription - //see 1/17/2014 for method//
2012 Field samples

=3/27/2014= qPCR DNased RNA - clean

=3/27/2014=

**Standard DNA-free treatment**
2012 Field samples

=3/26/2014=

**Standard DNA-free treatment - //see 1/16/2014 for method//**
2013 Field samples

=3/25/2014=

RNA extraction (part 2)
2012 Field samples

=3/24/2014=

RNA extraction (part 2) - //see 1/15/2014 for method//
2013 Field samples

=3/17/2014=

RNA extraction (part 1)
Homogenized 2013 Field P. herring liver samples (n=48), stored -80C -Samples from June, August, and October

=3/14/2014=

RNA extraction (part 1)
Homogenized 2012 Field P. herring liver samples (n=48), stored -80C -Samples from June, August, and October

= 2/27/2014 = qPCR - cDNA 'Time-course' samples 24-46 (20ul rxn) HIF-1a & EF-1a

23 samples 23 duplicates 2 NTC 2 error = 50 total rxns

cDNA template = 1ul

__//Master Mix// // HIF-1a P2-1 //____ //:// __ 2x Sso Fast Evogreen 10 x 50 = 500ul Forward primer 2-1 0.5 x 50 = 25ul Reverse primer 2-1 0.5 x 50 = 25ul H20 DEPC 0.1% 8 x 50 = 400ul

__//Master Mix EF////-1a P2//____//://__ 2x Sso Fast Evogreen 10 x 50 = 500ul Forward primer 2 0.5 x 50 = 25ul Reverse primer 2 0.5 x 50 = 25ul H20 DEPC 0.1% 8 x 50 = 400ul

qPCR - cDNA 'Time-course' samples 1-23 (20ul rxn) HIF-1a & EF-1a

23 samples 23 duplicates 2 NTC 2 error = 50 total rxns

cDNA template = 1ul

__ //Master Mix// //HIF-1a P2-1// ____ //:// __ 2x Sso Fast Evogreen 10 x 50 = 500ul Forward primer 2-1 0.5 x 50 = 25ul Reverse primer 2-1 0.5 x 50 = 25ul H20 DEPC 0.1% 8 x 50 = 400ul

__//Master Mix EF////-1a P2//____//://__ 2x Sso Fast Evogreen 10 x 50 = 500ul Forward primer 2 0.5 x 50 = 25ul Reverse primer 2 0.5 x 50 = 25ul H20 DEPC 0.1% 8 x 50 = 400ul

Reverse Transcription - TCL 101-144 44 samples 3 error = 47 rxns
 * 2/26/2014 **

Mean amount = 1194.46ug/ul Mean 260/280 = 1.71 Mean 260/230 = 1.65

Reverse Transcription - TCL 51-100 50 samples 4 error = 54 rxns
 * 2/25/2014 **

Mean amount = 1334.42ug/ul Mean 260/280 = 1.69 Mean 260/230 = 1.74

Reverse Transcription - TCL 1-50 50 samples 4 NTC = 54 rxns
 * 2/19/2014 **

Mean amount = 1433 ug/ul Mean 260/280 = 1.69 Mean 260/230 = 1.69

qPCR Time-course DNased RNA 51-144 94 samples 1 gDNA 1 NTC = 96 rxns
 * 2/18/2014 **

Results: Clean (i.e., no amplification)

qPCR Time-course DNased RNA 1-50 50 samples 1 gDNA 2 NTC = 53 rxns
 * 2/17/2014 **

Results: Clean (i.e., no amplification)

DNased RNA Time-course Sample 101-144
 * 2/13/2014 **

DNased RNATime-course Sample 51-100
 * 2/12/2014 **


 * 2/10/2014 **
 * Nandrop Time-course (Sample 1-50) __DNased__ RNA: **
 * Sample ID || ng/ul || 260/280 || 260/230 ||
 * TCL-1 || 286.71 || 1.97 || 1.43 ||
 * TCL-2 || 155.6 || 1.95 || 1.38 ||
 * TCL-3 || 153.5 || 1.96 || 1.01 ||
 * TCL-4 || 165.52 || 1.92 || 1.38 ||
 * TCL-5 || 163.01 || 1.97 || 1.42 ||
 * TCL-6 || 141.96 || 1.96 || 1.37 ||
 * TCL-7 || 90.09 || 1.89 || 1.13 ||
 * TCL-8 || 151.42 || 1.96 || 1.53 ||
 * TCL-9 || 158.92 || 1.95 || 1.37 ||
 * TCL-10 || 100.03 || 1.9 || 1.31 ||
 * TCL-11-2 || 181.95 || 1.83 || 1.03 ||
 * TCL-12 || 168.29 || 1.95 || 1.43 ||
 * TCL-13 || 103.82 || 1.92 || 1.37 ||
 * TCL-14 || 121.29 || 1.95 || 1.1 ||
 * TCL-15 || 150 || 1.94 || 0.62 ||
 * TCL-16 || 152.08 || 1.95 || 1.36 ||
 * TCL-17 || 106.98 || 1.9 || 1.31 ||
 * TCL-18 || 110.82 || 1.93 || 1.53 ||
 * TCL-19 || 165.86 || 1.95 || 1.43 ||
 * TCL-20 || 160.11 || 1.94 || 1.36 ||
 * TCL-21 || 190.23 || 1.96 || 1.65 ||
 * TCL-22 || 195.42 || 1.95 || 1.6 ||
 * TCL-23 || 129.87 || 1.93 || 1.17 ||
 * TCL-24 || 151.68 || 1.95 || 1.34 ||
 * TCL-25 || 153.89 || 1.91 || 1.54 ||
 * TCL-26 || 147.35 || 1.91 || 0.81 ||
 * TCL-27 || 160.08 || 1.96 || 1.41 ||
 * TCL-28 || 157.32 || 1.97 || 0.81 ||
 * TCL-29 || 90.66 || 1.93 || 1.43 ||
 * TCL-30 || 153.7 || 1.93 || 1.38 ||
 * TCL-31 || 117.77 || 1.91 || 0.97 ||
 * TCL-32 || 167.99 || 1.96 || 1.63 ||
 * TCL-33 || 134.84 || 1.9 || 1 ||
 * TCL-34-2 || 151.77 || 1.98 || 1.33 ||
 * TCL-35 || 109.33 || 1.91 || 1.31 ||
 * TCL-36 || 133.69 || 1.91 || 1.13 ||
 * TCL-37 || 88.72 || 1.91 || 1.36 ||
 * TCL-38 || 135.12 || 1.91 || 1.53 ||
 * TCL-39 || 106.91 || 1.94 || 1.36 ||
 * TCL-40 || 136.95 || 1.94 || 1.48 ||
 * TCL-41 || 161.95 || 1.96 || 1.58 ||
 * TCL-42 || 82.24 || 1.89 || 1.27 ||
 * TCL-43 || 121.64 || 1.96 || 1.55 ||
 * TCL-44 || 86.75 || 1.9 || 1.22 ||
 * TCL-45 || 111.54 || 1.93 || 1.23 ||
 * TCL-46 || 158.21 || 1.96 || 1.52 ||
 * TCL-47 || 107.82 || 1.9 || 1.23 ||
 * TCL-48 || 153.1 || 1.96 || 1.57 ||
 * TCL-49 || 226.39 || 1.97 || 1.23 ||
 * TCL-50 || 120.6 || 1.94 || 1.54 ||


 * 1/31/2014 **
 * Nandrop Time-course (Sample 51-100) RNA (pre-DNase): **


 * Sample || 260/280 || 260/230 || ng/ul ||
 * TCL 51 || 1.96 || 1.94 || 707.7 ||
 * TCL 52 || 2.01 || 1.79 || 1022.5 ||
 * TCL 53 || 1.97 || 2.01 || 788.1 ||
 * TCL 54 || 1.95 || 1.82 || 636.2 ||
 * TCL 55 || 1.95 || 1.98 || 616.9 ||
 * TCL 56 || 1.84 || 2.06 || 435.5 ||
 * TCL 57 || 1.97 || 2.06 || 716.4 ||
 * TCL 58 || 1.86 || 1.71 || 285.9 ||
 * TCL 59 || 1.77 || 1.36 || 150.3 ||
 * TCL 60 || 1.98 || 1.95 || 763.7 ||
 * TCL 61 || 1.98 || 1.75 || 700.9 ||
 * TCL 62 || 2 || 2.07 || 1035.6 ||
 * TCL 63 || 1.86 || 2.02 || 305 ||
 * TCL 64 || 1.87 || 1.83 || 394.9 ||
 * TCL 65 || 1.98 || 1.95 || 788.2 ||
 * TCL 66 || 1.88 || 1.92 || 319.2 ||
 * TCL 67 || 1.71 || 1.79 || 115.6 ||
 * TCL 68 || 1.96 || 1.99 || 678.3 ||
 * TCL 69 || 1.99 || 1.93 || 1217 ||
 * TCL 70 || 2 || 2..16 || 1102.6 ||
 * TCL 71 || 1.97 || 2.02 || 773 ||
 * TCL 72 || 1.93 || 2.07 || 644.5 ||
 * TCL 73 || 2.01 || 2.11 || 1204.7 ||
 * TCL 74 || 1.85 || 1.97 || 442.6 ||
 * TCL 75 || 1.96 || 1.75 || 634.1 ||
 * TCL 76 || 1.95 || 2 || 752.7 ||
 * TCL 77 || 2 || 2.12 || 1381 ||
 * TCL 78 || 1.98 || 1.98 || 866.3 ||
 * TCL 79 || 1.78 || 1.88 || 419.6 ||
 * TCL 80 || 2 || 1.8 || 855.6 ||
 * TCL 81 || 1.87 || 1.62 || 436.1 ||
 * TCL 82 || 2 || 1.93 || 1236.8 ||
 * TCL 83 || 1.94 || 2.06 || 735.2 ||
 * TCL 84 || 2 || 1.89 || 118.2 ||
 * TCL 85 || 1.88 || 1.66 || 243 ||
 * TCL 86 || 2 || 1.96 || 1026.6 ||
 * TCL 87 || 2 || 2.12 || 1640.9 ||
 * TCL 88 || 1.88 || 1.78 || 413.6 ||
 * TCL 89 || 1.89 || 1.8 || 383.2 ||
 * TCL 90 || 1.77 || 1.36 || 139.3 ||
 * TCL 91 || 1.94 || 2.04 || 651.6 ||
 * TCL 92 || 2 || 2.19 || 1963.9 ||
 * TCL 93 || 1.97 || 2.17 || 833.7 ||
 * TCL 94 || 1.97 || 2.11 || 791.8 ||
 * TCL 95 || 2 || 2.11 || 1101.9 ||
 * TCL 96 || 1.99 || 1.93 || 916.6 ||
 * TCL 97 || 2 || 2.14 || 1104.8 ||
 * TCL 98 || 1.86 || 1.76 || 257.6 ||
 * TCL 99 || 1.98 || 2.17 || 850.2 ||
 * TCL 100 || 2 || 2.22 || 1758.3 ||


 * 1/30/2014 **
 * Nandrop Time-course (Sample 1-50) RNA (pre-DNase): **
 * Sample || 260/280 || 260/230 || ng/ul ||
 * TCL 1 || 2.06 || 1.61 || 179.3 ||
 * TCL 2 || 1.87 || 1.84 || 258.3 ||
 * TCL 3 || 1.87 || 1.9 || 261.1 ||
 * TCL 4 || 1.85 || 1.9 || 253.3 ||
 * TCL 5 || 1.89 || 1.71 || 421.9 ||
 * TCL 6 || 1.98 || 1.63 || 612.8 ||
 * TCL 7 || 2 || 1.57 || 574.3 ||
 * TCL 8 || 1.86 || 1.99 || 460 ||
 * TCL 9 || 1.9 || 1.68 || 332.8 ||
 * TCL 10 || 2 || 1.97 || 846.9 ||
 * TCL 11 || 1.63 || 0.65 || 50.9 ||
 * TCL 12 || 1.89 || 1.8 || 353.9 ||
 * TCL 13 || 1.97 || 2.07 || 677.4 ||
 * TCL 14 || 2 || 1.48 || 688.3 ||
 * TCL 15 || 1.91 || 0.78 || 242.4 ||
 * TCL 16 || 1.9 || 1.88 || 357.9 ||
 * TCL 17 || 1.99 || 1.99 || 654.3 ||
 * TCL 18 || 1.88 || 2.26 || 922.9 ||
 * TCL 19 || 1.73 || 1.83 || 343.1 ||
 * TCL 20 || 1.7 || 1.92 || 234.1 ||
 * TCL 21 || 176 || 2.06 || 393.3 ||
 * TCL 22 || 1.72 || 1.99 || 343.2 ||
 * TCL 23 || 1.7 || 1.8 || 175.2 ||
 * TCL 24 || 1.71 || 1.81 || 264.7 ||
 * TCL 25 || 1.71 || 1.91 || 337.1 ||
 * TCL 26 || 1.68 || 1.83 || 197.5 ||
 * TCL 27 || 1.72 || 2.11 || 373.1 ||
 * TCL 28 || 1.72 || 1.93 || 406.2 ||
 * TCL 29 || 1.79 || 2.07 || 666.3 ||
 * TCL 30 || 1.69 || 2.06 || 228.5 ||
 * TCL 31 || 1.79 || 1.81 || 686.2 ||
 * TCL 32 || 1.83 || 2.13 || 859 ||
 * TCL 33 || 1.66 || 1.56 || 175.3 ||
 * TCL 34 || 1.61 || 8 || 88.4 ||
 * TCL 35 || 1.79 || 1.97 || 685.8 ||
 * TCL 36 || 1.7 || 1.91 || 262.1 ||
 * TCL 37 || 1.81 || 2.21 || 798.2 ||
 * TCL 38 || 1.68 || 2.1 || 458 ||
 * TCL 39 || 1.8 || 2.12 || 604.5 ||
 * TCL 40 || 1.69 || 2.11 || 263.8 ||
 * TCL 41 || 1.72 || 2.19 || 291.9 ||
 * TCL 42 || 1.82 || 2.04 || 755.8 ||
 * TCL 43 || 2 || 2.16 || 1141.5 ||
 * TCL 44 || 2 || 2.08 || 912.9 ||
 * TCL 45 || 1.78 || 1.72 || 145.7 ||
 * TCL 46 || 1.98 || 1.93 || 636.1 ||
 * TCL 47 || 1.99 || 1.95 || 796.2 ||
 * TCL 48 || 1.97 || 2.01 || 607 ||
 * TCL 49 || 1.89 || 1.27 || 456.6 ||
 * TCL 50 || 2 || 2.25 || 997.3 ||

homogenized tissue samples stored -80C
 * 1/27 - 1/29/2014 **
 * RNA Extraction (Part 1) - Time-course P. herring liver tissue samples (TCL 1-144)**

qPCR - cDNA 'Threshold' Reps 2 & 3 (20ul rxn) HIF-1a & EF-1a
 * 1/22/2014 **


 * //Note: two rounds of qPCR were conducted separately for the HIF and EF genes//**

36 samples 36 duplicates 2 NTC 6 error = 80 total rxns

cDNA template = 1ul

__ //Master Mix:// __ 2x Sso Fast Evogreen 10 x 80 = 800ul Forward primer 2 0.5 x 80 = 40ul **//Used HIF-1a P2-1 & EF-1a P2//**
 * Reverse primer 2 0.5 x 80 = 40ul **
 * H20 DEPC 0.1% 8 x 82 = 656ul **

1/17/2014 & 1/21/2014 Reverse Transcription (Promega M-MLV Protocol)

RT the 'Threshold' lab samples (Replicates 2 & 3) to create cDNA


 * A single reaction volume =** 25uL**. The volume of RNA, primer(s) and M-MLV RT used are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA.

1) //Calculate volume of RNA = 1ug of RNA://

3) //Add appropriate amount of primer to sample. Use 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT in this example). Total volume (RNA + primers) should equal 18.25uL//.
 * **Sample** || **ng/ul** || **ug/ul** || **V (ul) of 1ug of RNA** || **DEPC 0.1%** || **Total V (ul)** ||
 * 31-H || 222.5 || 0.2225 || 4.5 || 13.3 || 17.75 ||
 * 32-H || 133.6 || 0.1336 || 7.5 || 10.3 || 17.75 ||
 * 33-H || 157.2 || 0.1572 || 6.4 || 11.4 || 17.75 ||
 * 34-H || 150.8 || 0.1508 || 6.6 || 11.1 || 17.75 ||
 * 35-H || 194.2 || 0.1942 || 5.1 || 12.6 || 17.75 ||
 * 36-H || 178.2 || 0.1782 || 5.6 || 12.1 || 17.75 ||
 * 31-M || 188.8 || 0.1888 || 5.3 || 12.5 || 17.75 ||
 * 32-M || 146.8 || 0.1468 || 6.8 || 10.9 || 17.75 ||
 * 33-M || 111.1 || 0.1111 || 9.0 || 8.7 || 17.75 ||
 * 34-M || 155 || 0.155 || 6.5 || 11.3 || 17.75 ||
 * 35-M || 137.9 || 0.1379 || 7.3 || 10.5 || 17.75 ||
 * 36-M || 87.1 || 0.0871 || 11.5 || 6.3 || 17.75 ||
 * 31-C || 136.7 || 0.1367 || 7.3 || 10.4 || 17.75 ||
 * 32-C || 157 || 0.157 || 6.4 || 11.4 || 17.75 ||
 * 33-C || 143.2 || 0.1432 || 7.0 || 10.8 || 17.75 ||
 * 34-C || 154.1 || 0.1541 || 6.5 || 11.3 || 17.75 ||
 * 35-C || 177.2 || 0.1772 || 5.6 || 12.1 || 17.75 ||
 * 36-C || 119.9 || 0.1199 || 8.3 || 9.4 || 17.75 ||
 * 49-H || 164.5 || 0.1645 || 6.1 || 11.7 || 17.75 ||
 * 50-H || 142.5 || 0.1425 || 7.0 || 10.7 || 17.75 ||
 * 51-H || 91.8 || 0.0918 || 10.9 || 6.9 || 17.75 ||
 * 52-H || 179.1 || 0.1791 || 5.6 || 12.2 || 17.75 ||
 * 53-H || 170 || 0.17 || 5.9 || 11.9 || 17.75 ||
 * 54-H || 134.8 || 0.1348 || 7.4 || 10.3 || 17.75 ||
 * 49-M || 167.6 || 0.1676 || 6.0 || 11.8 || 17.75 ||
 * 50-M || 134.1 || 0.1341 || 7.5 || 10.3 || 17.75 ||
 * 51-M || 154.1 || 0.1541 || 6.5 || 11.3 || 17.75 ||
 * 52-M || 186.1 || 0.1861 || 5.4 || 12.4 || 17.75 ||
 * 53-M || 181.3 || 0.1813 || 5.5 || 12.2 || 17.75 ||
 * 54-M || 165.3 || 0.1653 || 6.0 || 11.7 || 17.75 ||
 * 49-C || 156.9 || 0.1569 || 6.4 || 11.4 || 17.75 ||
 * 50-C || 240.6 || 0.2406 || 4.2 || 13.6 || 17.75 ||
 * 51-C || 134.2 || 0.1342 || 7.5 || 10.3 || 17.75 ||
 * 52-C || 166.5 || 0.1665 || 6.0 || 11.7 || 17.75 ||
 * 53-C || 247.6 || 0.2476 || 4.0 || 13.7 || 17.75 ||
 * 54-C || 153.5 || 0.1535 || 6.5 || 11.2 || 17.75 ||

17.75ulRNA + 0.5ul Promega oligo dT = 18.25ul

4) //Heat samples at 70C for 5 min in thermocycler.// 5) // Placed samples on ice IMMEDIATELY. // 6) //Made __**Master Mix** (40 RXNs)__//

__PER RXN__

5 uL 5x Buffer (M-MLV RT Buffer) x 40 = 200ul

1.25 uL 2.5mM dNTPs x 40 = 50ul

0.5 uL M-MLV RT **per ug of RNA** x 40 **=** 20ul

//7) Mix well.// //8) Added 6.75uL of master mix to each reaction.// //9) Mix well, flicked lightly// //10) Spot spun// //11) Incubate @ 42C for 1hr in thermalcycler for oligo dT primers OR @ 37C for random primers.// //12) Heat inactivate @ 95C for 3 min.// //13) Spot spun.//

1. Each RNA sample was diluted to equal 10ug of RNA for 50ul rxn in a **0.5mL tube**: 2. **5ul** of the **TURBO** **DNase** **buffer** to each sample 3. **1ul** of **TURBO** **DNase** was added to each sample 4. Sample were incubated for 30min @ 37C 6. DNase Inactivation Reagent was resuspended (flicked) 7. **5ul** of the **Inactivation** **Reagent** was added to each sample 8. Samples incubated for ~2min @ RT, each mixed/flicked an additional time to resuspend the reagent 9. Samples centrifuge at 10,000 x g for 1.5min 10. Supernatent was carefully pipetted to new (labeled) 0.5mL tubes.
 * 1/16/2014 **
 * Standard DNA-free treatment: **


 * **Sample** || **ng/ul** || **ug/ul** || **Dilute = 10ug RNA = V ul of RNA** || **DEPC 0.1%** || **Total V (ul)** || **Turbo Dnase Buffer (0.1 V ul)** || **Turbo Dnase (ul)** || **DNase Inactivetion Reagent (ul)** ||
 * 31-H || 342 || 0.342 || 29.24 || 20.76 || 50.00 || 5.00 || 1.00 || 5 ||
 * 32-H || 308 || 0.308 || 32.47 || 17.53 || 50.00 || 5.00 || 1.00 || 5 ||
 * 33-H || 328.4 || 0.3284 || 30.45 || 19.55 || 50.00 || 5.00 || 1.00 || 5 ||
 * 34-H || 621.9 || 0.6219 || 16.08 || 33.92 || 50.00 || 5.00 || 1.00 || 5 ||
 * 35-H || 422.8 || 0.4228 || 23.65 || 26.35 || 50.00 || 5.00 || 1.00 || 5 ||
 * 36-H || 329.2 || 0.3292 || 30.38 || 19.62 || 50.00 || 5.00 || 1.00 || 5 ||
 * 31-M || 216.3 || 0.2163 || 46.23 || 3.77 || 50.00 || 5.00 || 1.00 || 5 ||
 * 32-M || 1104.3 || 1.1043 || 9.06 || 40.94 || 50.00 || 5.00 || 1.00 || 5 ||
 * 33-M || 145.5 || 0.1455 || 50.00 || 0.00 || 50.00 || 5.00 || 1.00 || 5 ||
 * 34-M || 113.6 || 0.1136 || 50.00 || 0.00 || 50.00 || 5.00 || 1.00 || 5 ||
 * 35-M || 792.8 || 0.7928 || 12.61 || 37.39 || 50.00 || 5.00 || 1.00 || 5 ||
 * 36-M || 137.6 || 0.1376 || 50.00 || 0.00 || 50.00 || 5.00 || 1.00 || 5 ||
 * 31-C || 1033.6 || 1.0336 || 9.67 || 40.33 || 50.00 || 5.00 || 1.00 || 5 ||
 * 32-C || 306.1 || 0.3061 || 32.67 || 17.33 || 50.00 || 5.00 || 1.00 || 5 ||
 * 33-C || 303.4 || 0.3034 || 32.96 || 17.04 || 50.00 || 5.00 || 1.00 || 5 ||
 * 34-C || 651.7 || 0.6517 || 15.34 || 34.66 || 50.00 || 5.00 || 1.00 || 5 ||
 * 35-C || 304.8 || 0.3048 || 32.81 || 17.19 || 50.00 || 5.00 || 1.00 || 5 ||
 * 36-C || 644.2 || 0.6442 || 15.52 || 34.48 || 50.00 || 5.00 || 1.00 || 5 ||
 * 49-H || 166.4 || 0.1664 || 50.00 || 0.00 || 50.00 || 5.00 || 1.00 || 5 ||
 * 50-H || 397.4 || 0.3974 || 25.16 || 24.84 || 50.00 || 5.00 || 1.00 || 5 ||
 * 51-H || 405.7 || 0.4057 || 24.65 || 25.35 || 50.00 || 5.00 || 1.00 || 5 ||
 * 52-H || 427.9 || 0.4279 || 23.37 || 26.63 || 50.00 || 5.00 || 1.00 || 5 ||
 * 53-H || 244.3 || 0.2443 || 40.93 || 9.07 || 50.00 || 5.00 || 1.00 || 5 ||
 * 54-H || 388.9 || 0.3889 || 25.71 || 24.29 || 50.00 || 5.00 || 1.00 || 5 ||
 * 49-M || 316.4 || 0.3164 || 31.61 || 18.39 || 50.00 || 5.00 || 1.00 || 5 ||
 * 50-M || 641.5 || 0.6415 || 15.59 || 34.41 || 50.00 || 5.00 || 1.00 || 5 ||
 * 51-M || 360.3 || 0.3603 || 27.75 || 22.25 || 50.00 || 5.00 || 1.00 || 5 ||
 * 52-M || 444.9 || 0.4449 || 22.48 || 27.52 || 50.00 || 5.00 || 1.00 || 5 ||
 * 53-M || 332.3 || 0.3323 || 30.09 || 19.91 || 50.00 || 5.00 || 1.00 || 5 ||
 * 54-M || 283.7 || 0.2837 || 35.25 || 14.75 || 50.00 || 5.00 || 1.00 || 5 ||
 * 49-C || 384.9 || 0.3849 || 25.98 || 24.02 || 50.00 || 5.00 || 1.00 || 5 ||
 * 50-C || 1020.7 || 1.0207 || 9.80 || 40.20 || 50.00 || 5.00 || 1.00 || 5 ||
 * 51-C || 649.6 || 0.6496 || 15.39 || 34.61 || 50.00 || 5.00 || 1.00 || 5 ||
 * 52-C || 282.8 || 0.2828 || 35.36 || 14.64 || 50.00 || 5.00 || 1.00 || 5 ||
 * 53-C || 217.1 || 0.2171 || 46.06 || 3.94 || 50.00 || 5.00 || 1.00 || 5 ||
 * 54-C || 374.1 || 0.3741 || 26.73 || 23.27 || 50.00 || 5.00 || 1.00 || 5 ||

qPCR 'clean' RNA 'Threshold' Reps 1 & 4 (20ul rxn) 36 samples 3 NTC 1 gDNA 4 error = 43 total rxns

RNA template = 0.5ul gDNA template = 0.5ul

__//Master Mix://__ //*Note: used EF-1a primer pair 1 that works but is not being used for normalization//

2x Sso Fast Evogreen 10 x 43 = 430ul

Forward EF-1a p1 0.5 x 43 = 21.5ul

Reverse EF-1a p1 0.5 x 43 = 21.5ul

H20 DEPC 0.1% 8.5 x 43 = 365.5ul

RESULTS:

**1/15/2014**

//**__RNA extraction continued (see 1.14.2014):__**//

1) Tubes incubated at RT for 5min

2) Under __**fume** **hood**__ 200ul of chloroform was added to each sample

3) Each sample vortexed vigorously for 30sec for 'milky' emulsion to occur

4) Incubated at RT for 5min

5) Tubes spun down for 15min, max speed, 4C

6) Tubes gently removed

7) Aqueous phase (top layer) carefully transferred to new 1.5ml tubes

8) 500ul of isopropanol added to new tube containing the RNA

9) Invert several time to mix

10) Incubated 10min at RT

11) Spun down max speed, 8min, 4C; tube hinge point up

12) Small white pellet present (RNA)

13) Supernatent removed

14) 1ml of 75% EtOH added to tube with pellet.

15) Vortex briefly to dislodge pellet

16) Spun down at 7500g for 5min

17) Carefully remove supernatent

18) Briefly spun down again (~15s) to pool residual EtOH

19) EtOH removed with P10 pipette

20) Tubes left open for no more than 5min

21) Pellets re-suspended with 100ul of 0.1% DEPC-H2O

22) Tubes incubated at 55C for 5min (waterbath)

23) Tubes flicked several times

24) Stored at -80C

qPCR Normalizing gene (EF-1a) - cDNA 'Threshold' Reps 1 & 4 (20ul rxn) 36 samples 36 duplicates 3 NTC 8 error = 82 total rxns

cDNA template = 1ul

__//Master Mix://__

2x Sso Fast Evogreen 10 x 82 = 820ul

Forward 2-1 0.5 x 82 = 41ul

Reverse 2-1 0.5 x 82 = 41ul

H20 DEPC 0.1% 8 x 82 = 656ul


 * RESULTS: ([[file:20140114_113921_Halley_EF-1a(p2).tad|20140114_113921_Halley_EF-1a(p2).tad]])**



__**RNA Extraction**__ (TriReagent) of 'Threshold' experimental samples from MMFS: Replicates 2 & 3

Rep 2: 31-H to 36-H; 31-M to 36-M; 31-C to 36-C

Rep 3: 49-H to 54- H; 49-M to 54-M; 39-C to 54-C

1) 500ul of TriReagent added to each sample in a 1.5ml snap-cap tube

2) Each sample homogenized with sterile pestle

3) An additional 500ul added

4) Each sample vortexed vigorously for 15s

5) Stored at -80C

7/31/2013
cDNA nanodrop
 * Sample || ng/ul || 260/280 || 260/230 ||
 * 13-H || 222 || 1.76 || 1.95 ||
 * 14-H || 309.7 || 1.8 || 2 ||
 * 15-H || 221.4 || 1.78 || 2.03 ||
 * 16-H || 301.1 || 1.78 || 2.05 ||
 * 17-H || 415.7 || 1.78 || 2.02 ||
 * 18-H || 368.3 || 1.77 || 2.05 ||
 * 67-H || 346.3 || 1.39 || 1.49 ||
 * 68-H || 294.5 || 1.34 || 1.4 ||
 * 69-H || 283.9 || 1.4 || 1.51 ||
 * 70-H || 263.4 || 1.41 || 1.54 ||
 * 71-H || 332.3 || 1.33 || 1.3 ||
 * 72-H || 232.5 || 1.54 || 1.56 ||
 * 72.5-H || 308.2 || 1.35 || 1.45 ||
 * 13-M || 296 || 1.32 || 1.39 ||
 * 14-M || 290.8 || 1.32 || 1.41 ||
 * 15-M || 318.9 || 1.37 || 1.49 ||
 * 16-M || 309.1 || 1.33 || 1.35 ||
 * 17-M || 296.8 || 1.34 || 1.45 ||
 * 18-M || 286.9 || 1.33 || 1.44 ||
 * 67-M || 297.2 || 1.31 || 1.2 ||
 * 68-M || 294.3 || 1.31 || 1.44 ||
 * 69-M || 282.1 || 1.31 || 1.46 ||
 * 70-M || 310.1 || 1.3 || 1.28 ||
 * 71-M || 281.4 || 1.32 || 1.46 ||
 * 72-M || 326.1 || 1.33 || 1.25 ||
 * 13-C || 311 || 1.32 || 1.45 ||
 * 14-C || 311.5 || 1.31 || 1.44 ||
 * 15-C || 306.3 || 1.32 || 1.3 ||
 * 16-C || 291 || 1.3 || 1.47 ||
 * 17-C || 284.2 || 1.31 || 1.4 ||
 * 18-C || 286.3 || 1.31 || 1.46 ||
 * 67-C || 309.5 || 1.34 || 1.37 ||
 * 68-C || 316.6 || 1.32 || 1.27 ||
 * 69-C || 301.5 || 1.31 || 1.29 ||
 * 70-C || 309.8 || 1.35 || 1.45 ||
 * 71-C || 280.1 || 1.32 || 1.45 ||
 * 72-C || 254.8 || 1.3 || 2.1 ||

qPCR - cDNA 'Threshold' Reps 1 & 4 (20ul rxn) 37 samples 37 duplicates 2 NTC 4 error = 80 total rxns

cDNA template = 1ul

__//Master Mix://__

2x Sso Fast Evogreen 10 x 80 = 800ul

Forward 2-1 0.5 x 80 = 40ul

Reverse 2-1 0.5 x 80 = 40ul

H20 DEPC 0.1% 8 x 80 = 640ul

Results:

7/30/2013

 * Reverse Transcription (Promega M-MLV Protocol) **


 * RT the 'Threshold' lab samples (Replicates 1 & 4) to create cDNA**

A single reaction volume = **25uL**. The volume of RNA, primer(s) and M-MLV RT used are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.

1) //Calculate volume of RNA = 1ug of RNA://


 * Sample || ng/ul || ug/ul || V (ul) of RNA = 1ug || DEPC 0.1% (ul) || Total Rxn ||
 * 13-H || 133.4 || 0.1334 || 7.50 || 10.3 || 17.75 ||
 * 14-H || 113.4 || 0.1134 || 8.82 || 8.9 || 17.75 ||
 * 15-H || 147.4 || 0.1474 || 6.78 || 11.0 || 17.75 ||
 * 16-H || 157.4 || 0.1574 || 6.35 || 11.4 || 17.75 ||
 * 17-H || 133.1 || 0.1331 || 7.51 || 10.2 || 17.75 ||
 * 18-H || 145.5 || 0.1455 || 6.87 || 10.9 || 17.75 ||
 * 67-H || 103.9 || 0.1039 || 9.62 || 8.1 || 17.75 ||
 * 68-H || 140.2 || 0.1402 || 7.13 || 10.6 || 17.75 ||
 * 69-H || 108.3 || 0.1083 || 9.23 || 8.5 || 17.75 ||
 * 70-H || 89.7 || 0.0897 || 11.15 || 6.6 || 17.75 ||
 * 71-H || 136.4 || 0.1364 || 7.33 || 10.4 || 17.75 ||
 * 72-H || 187.2 || 0.1872 || 5.34 || 12.4 || 17.75 ||
 * 72.5-H || 132.2 || 0.1322 || 7.56 || 10.2 || 17.75 ||
 * 13-M || 132.3 || 0.1323 || 7.56 || 10.2 || 17.75 ||
 * 14-M || 142.1 || 0.1421 || 7.04 || 10.7 || 17.75 ||
 * 15-M || 148.7 || 0.1487 || 6.72 || 11.0 || 17.75 ||
 * 16-M || 144.1 || 0.1441 || 6.94 || 10.8 || 17.75 ||
 * 17-M || 167.7 || 0.1677 || 5.96 || 11.8 || 17.75 ||
 * 18-M || 174.4 || 0.1744 || 5.73 || 12.0 || 17.75 ||
 * 67-M || 155.7 || 0.1557 || 6.42 || 11.3 || 17.75 ||
 * 68-M || 97.9 || 0.0979 || 10.21 || 7.5 || 17.75 ||
 * 69-M || 175.4 || 0.1754 || 5.70 || 12.0 || 17.75 ||
 * 70-M || 201.5 || 0.2015 || 4.96 || 12.8 || 17.75 ||
 * 71-M || 157.1 || 0.1571 || 6.37 || 11.4 || 17.75 ||
 * 72-M || 152.4 || 0.1524 || 6.56 || 11.2 || 17.75 ||
 * 13-C || 139.3 || 0.1393 || 7.18 || 10.6 || 17.75 ||
 * 14-C || 135.1 || 0.1351 || 7.40 || 10.3 || 17.75 ||
 * 15-C || 140.4 || 0.1404 || 7.12 || 10.6 || 17.75 ||
 * 16-C || 152.5 || 0.1525 || 6.56 || 11.2 || 17.75 ||
 * 17-C || 169 || 0.169 || 5.92 || 11.8 || 17.75 ||
 * 18-C || 140.2 || 0.1402 || 7.13 || 10.6 || 17.75 ||
 * 67-C || 155.03 || 0.15503 || 6.45 || 11.3 || 17.75 ||
 * 68-C || 180 || 0.18 || 5.56 || 12.2 || 17.75 ||
 * 69-C || 146.8 || 0.1468 || 6.81 || 10.9 || 17.75 ||
 * 70-C || 150.5 || 0.1505 || 6.64 || 11.1 || 17.75 ||
 * 71-C || 141.7 || 0.1417 || 7.06 || 10.7 || 17.75 ||
 * 72-C || 163 || 0.163 || 6.13 || 11.6 || 17.75 ||

3) //Add appropriate amount of primer to sample. Use 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT in this example). Total volume (RNA + primers) should equal 18.25uL//.

17.75ulRNA + 0.5ul Promega oligo dT = 18.25ul

4) // Heat samples at 70C for 5 min in thermocycler. //

5) //Placed samples on ice IMMEDIATELY.//

6) //Made __**Master Mix** (40 RXNs)__//

__PER RXN__

5 uL 5x Buffer (M-MLV RT Buffer) x 40 = 200ul

1.25 uL 2.5mM dNTPs x 40 = 50ul

0.5 uL M-MLV RT **per ug of RNA** x 40 **=** 20ul

//7) Mix well.//

//8) Added 6.75uL of master mix to each reaction.//

//9) Mix well, flicked lightly//

//10) Spot spun//

//11) Incubate @ 42C for 1hr in thermalcycler for oligo dT primers OR @ 37C for random primers.//

//12) Heat inactivate @ 95C for 3 min.//

//13) Spot spun.//

//14)Stored @ -20C.//

7/26/2013
qPCR - DNased RNA 'Threshold' Reps 1 & 4

36 samples 1 gDNA 2 NTC 1 error

= 40 total rxns

__//Master Mix://__ 2x Sso Fast Evogreen 10 x 40 = 400ul Forward 2-1 0.5 x 40 = 20ul Reverse 2-1 0.5 x 40 = 20ul H20 DEPC 0.1% 8.5 x 40 = 340ul

Results: RNA clean (no amplification)

7/25/2013
Nanodrop RNA:

Standard DNA-free treatment: 1. Each RNA sample was diluted to equal 10ug of RNA for 50ul rxn in a **0.5mL tube**: 2. 5ul of the TURBO DNase buffer to each sample 3. 1ul of TURBO DNase was added to each sample 4. Sample were incubated for 30min @ 37C 6. DNase Inactivation Reagent was resuspended (flicked) 7. 5ul of the Inactivation Reagent was added to each sample 8. Samples incubated for ~2min @ RT, each mixed/flicked an additional time to resuspend the reagent 9. Samples centrifuge at 10,000 x g for 1.5min 10. Supernatent was carefully pipetted to new (labeled) 0.5mL tubes.
 * Sample || ng/ul || 260/280 || 260/230 ||
 * 13-H || 656.8 || 2.07 || 2.09 ||
 * 14-H || 787.3 || 2.02 || 2.33 ||
 * 15-H || 1021.7 || 2.05 || 2.07 ||
 * 16-H || 942.8 || 2.05 || 2.18 ||
 * 17-H || 1516.3 || 2.11 || 2.13 ||
 * 18-H || 660.8 || 2.05 || 1.87 ||
 * 67-H || 327.6 || 2 || 1.84 ||
 * 68-H || 367.7 || 2 || 1.82 ||
 * 69-H || 1526.9 || 2.1 || 2.23 ||
 * 70-H || 597 || 2.03 || 2.17 ||
 * 71-H || 565.2 || 2.08 || 1.47 ||
 * 72-H || 707.3 || 2.08 || 1.81 ||
 * 72.5-H || 576.7 || 2.05 || 1.79 ||
 * 13-M || 817.9 || 2.08 || 1.89 ||
 * 14-M || 588.4 || 2.06 || 1.68 ||
 * 15-M || 386.34 || 1.94 || 1.95 ||
 * 16-M || 210.2 || 1.9 || 2 ||
 * 17-M || 804.2 || 2.08 || 1.79 ||
 * 18-M || 381.3 || 1.98 || 1.92 ||
 * 67-M || 688.9 || 2.09 || 0.78 ||
 * 68-M || 948.9 || 2.08 || 2.14 ||
 * 69-M || 421.2 || 1.97 || 2.03 ||
 * 70-M || 926.3 || 2.07 || 2.13 ||
 * 71-M || 1042.2 || 2.07 || 2.02 ||
 * 72-M || 384 || 1.95 || 2.14 ||
 * 13-C || 730.4 || 2.05 || 1.95 ||
 * 14-C || 645.1 || 2.07 || 1.47 ||
 * 15-C || 841 || 2.04 || 2.02 ||
 * 16-C || 773.5 || 2.06 || 1.95 ||
 * 17-C || 455.8 || 1.96 || 1.91 ||
 * 18-C || 940.4 || 2.07 || 2.08 ||
 * 67-C || 1207 || 2.1 || 1.51 ||
 * 68-C || 459.7 || 1.97 || 1.9 ||
 * 69-C || 680.2 || 2.06 || 1.8 ||
 * 70-C || 809.2 || 2.07 || 1.71 ||
 * 71-C || 772.8 || 2.07 || 1.79 ||
 * 72-C || 293 || 197 || 1.96 ||


 * Sample || ng/ul || ug/ul || Dilute = 10ug RNA = V ul of RNA || DEPC 0.1% || Total (ul) || Turbo Dnase Buffer (ul) || Turbo Dnase (ul) || DNase Inactivetion Reagent (ul) ||
 * 13-H || 656.8 || 0.6568 || 15.23 || 34.77 || 50.00 || 5.00 || 1.00 || 5 ||
 * 14-H || 787.3 || 0.7873 || 12.70 || 37.30 || 50.00 || 5.00 || 1.00 || 5 ||
 * 15-H || 1021.7 || 1.0217 || 9.79 || 40.21 || 50.00 || 5.00 || 1.00 || 5 ||
 * 16-H || 942.8 || 0.9428 || 10.61 || 39.39 || 50.00 || 5.00 || 1.00 || 5 ||
 * 17-H || 1516.3 || 1.5163 || 6.60 || 43.40 || 50.00 || 5.00 || 1.00 || 5 ||
 * 18-H || 660.8 || 0.6608 || 15.13 || 34.87 || 50.00 || 5.00 || 1.00 || 5 ||
 * 67-H || 327.6 || 0.3276 || 30.53 || 19.47 || 50.00 || 5.00 || 1.00 || 5 ||
 * 68-H || 367.7 || 0.3677 || 27.20 || 22.80 || 50.00 || 5.00 || 1.00 || 5 ||
 * 69-H || 1526.9 || 1.5269 || 6.55 || 43.45 || 50.00 || 5.00 || 1.00 || 5 ||
 * 70-H || 597 || 0.597 || 16.75 || 33.25 || 50.00 || 5.00 || 1.00 || 5 ||
 * 71-H || 565.2 || 0.5652 || 17.69 || 32.31 || 50.00 || 5.00 || 1.00 || 5 ||
 * 72-H || 707.3 || 0.7073 || 14.14 || 35.86 || 50.00 || 5.00 || 1.00 || 5 ||
 * 72.5-H || 576.7 || 0.5767 || 17.34 || 32.66 || 50.00 || 5.00 || 1.00 || 5 ||
 * 13-M || 817.9 || 0.8179 || 12.23 || 37.77 || 50.00 || 5.00 || 1.00 || 5 ||
 * 14-M || 588.4 || 0.5884 || 17.00 || 33.00 || 50.00 || 5.00 || 1.00 || 5 ||
 * 15-M || 386.34 || 0.38634 || 25.88 || 24.12 || 50.00 || 5.00 || 1.00 || 5 ||
 * 16-M || 210.2 || 0.2102 || 47.57 || 2.43 || 50.00 || 5.00 || 1.00 || 5 ||
 * 17-M || 804.2 || 0.8042 || 12.43 || 37.57 || 50.00 || 5.00 || 1.00 || 5 ||
 * 18-M || 381.3 || 0.3813 || 26.23 || 23.77 || 50.00 || 5.00 || 1.00 || 5 ||
 * 67-M || 688.9 || 0.6889 || 14.52 || 35.48 || 50.00 || 5.00 || 1.00 || 5 ||
 * 68-M || 948.9 || 0.9489 || 10.54 || 39.46 || 50.00 || 5.00 || 1.00 || 5 ||
 * 69-M || 421.2 || 0.4212 || 23.74 || 26.26 || 50.00 || 5.00 || 1.00 || 5 ||
 * 70-M || 926.3 || 0.9263 || 10.80 || 39.20 || 50.00 || 5.00 || 1.00 || 5 ||
 * 71-M || 1042.2 || 1.0422 || 9.60 || 40.40 || 50.00 || 5.00 || 1.00 || 5 ||
 * 72-M || 384 || 0.384 || 26.04 || 23.96 || 50.00 || 5.00 || 1.00 || 5 ||
 * 13-C || 730.4 || 0.7304 || 13.69 || 36.31 || 50.00 || 5.00 || 1.00 || 5 ||
 * 14-C || 645.1 || 0.6451 || 15.50 || 34.50 || 50.00 || 5.00 || 1.00 || 5 ||
 * 15-C || 841 || 0.841 || 11.89 || 38.11 || 50.00 || 5.00 || 1.00 || 5 ||
 * 16-C || 773.5 || 0.7735 || 12.93 || 37.07 || 50.00 || 5.00 || 1.00 || 5 ||
 * 17-C || 455.8 || 0.4558 || 21.94 || 28.06 || 50.00 || 5.00 || 1.00 || 5 ||
 * 18-C || 940.4 || 0.9404 || 10.63 || 39.37 || 50.00 || 5.00 || 1.00 || 5 ||
 * 67-C || 1207 || 1.207 || 8.29 || 41.71 || 50.00 || 5.00 || 1.00 || 5 ||
 * 68-C || 459.7 || 0.4597 || 21.75 || 28.25 || 50.00 || 5.00 || 1.00 || 5 ||
 * 69-C || 680.2 || 0.6802 || 14.70 || 35.30 || 50.00 || 5.00 || 1.00 || 5 ||
 * 70-C || 809.2 || 0.8092 || 12.36 || 37.64 || 50.00 || 5.00 || 1.00 || 5 ||
 * 71-C || 772.8 || 0.7728 || 12.94 || 37.06 || 50.00 || 5.00 || 1.00 || 5 ||
 * 72-C || 293 || 0.293 || 34.13 || 15.87 || 50.00 || 5.00 || 1.00 || 5 ||

"Clean" RNA was then spec'ed in the Nano-Drop:


 * Sample || ng/ul || 260/280 || 260/230 ||
 * 13-H || 133.4 || 2.01 || 1.53 ||
 * 14-H || 113.4 || 1.95 || 1.65 ||
 * 15-H || 147.4 || 1.93 || 1.67 ||
 * 16-H || 157.4 || 1.99 || 1.69 ||
 * 17-H || 133.1 || 1.97 || 1.66 ||
 * 18-H || 145.5 || 1.94 || 1.47 ||
 * 67-H || 103.9 || 1.96 || 1.31 ||
 * 68-H || 140.2 || 1.95 || 1.33 ||
 * 69-H || 108.3 || 2.01 || 1.61 ||
 * 70-H || 89.7 || 1.98 || 1.47 ||
 * 71-H || 136.4 || 1.94 || 1.35 ||
 * 72-H || 187.2 || 1.98 || 1.69 ||
 * 72.5-H || 132.2 || 1.97 || 1.44 ||
 * 13-M || 132.3 || 2 || 1.62 ||
 * 14-M || 142.1 || 1.97 || 1.45 ||
 * 15-M || 148.7 || 1.97 || 1.6 ||
 * 16-M || 144.1 || 1.95 || 1.54 ||
 * 17-M || 167.7 || 1.99 || 1.5 ||
 * 18-M || 174.4 || 1.96 || 1.49 ||
 * 67-M || 155.7 || 2.01 || 0.76 ||
 * 68-M || 97.9 || 1.98 || 1.46 ||
 * 69-M || 175.4 || 1.97 || 1.69 ||
 * 70-M || 201.5 || 1.98 || 1.66 ||
 * 71-M || 157.1 || 1.96 || 1.63 ||
 * 72-M || 152.4 || 1.94 || 1.6 ||
 * 13-C || 139.3 || 1.99 || 1.55 ||
 * 14-C || 135.1 || 1.99 || 1.44 ||
 * 15-C || 140.4 || 2 || 1.64 ||
 * 16-C || 152.5 || 1.98 || 1.54 ||
 * 17-C || 169 || 1.99 || 1.4 ||
 * 18-C || 140.2 || 1.98 || 1.63 ||
 * 67-C || 155.03 || 1.98 || 1.20 ||
 * 68-C || 180 || 1.99 || 1.64 ||
 * 69-C || 146.8 || 1.97 || 1.14 ||
 * 70-C || 150.5 || 1.98 || 1.62 ||
 * 71-C || 141.7 || 1.98 || 1.6 ||
 * 72-C || 163 || 1.99 || 1.56 ||

//RNA stored at -80C//

**7/24/2013** //**__RNA extraction continued:__**// 1) Tubes incubated at RT for 5min 2) Under __**fume** **hood**__ 200ul of chloroform was added to each sample 3) Each sample vortexed vigorously for 30sec for 'milky' emulsion to occur 4) Incubated at RT for 5min 5) Tubes spun down for 15min, max speed, 4C 6) Tubes gently removed 7) Aqueous phase (top layer) carefully transferred to new 1.5ml tubes 8) 500ul of isopropanol added to new tube containing the RNA 9) Invert several time to mix 10) Incubated 10min at RT 11) Spun down max speed, 8min, 4C; tube hinge point up 12) Small white pellet present (RNA) 13) Supernatent removed 14) 1ml of 75% EtOH added to tube with pellet. 15) Vortex briefly to dislodge pellet 16) Spun down at 7500g for 5min 17) Carefully remove supernatent 18) Briefly spun down again (~15s) to pool residual EtOH 19) EtOH removed with P10 pipette 20) Tubes left open for no more than 5min 21) Pellets re-suspended with 100ul of 0.1% DEPC-H2O 22) Tubes incubated at 55C for 5min (waterbath) 23) Tubes flicked several times 24) Stored at -80C

7/23/2013
__**RNA Extraction**__ (TriReagent) of experimental samples from MMFS: Replicates 1 & 4

Rep 1: 13-H to 18-H; 13-M to 18-M; 13-C to 18-C Rep 4: 67-H to 72.5- H; 67-M to 72-M; 67-C to 72-C

1) 500ul of TriReagent added to each sample in a 1.5ml snap-cap tube 2) Each sample homogenized with sterile pestle 3) An additional 500ul added 4) Each sample vortexed vigorously for 15s 5) Stored at -80C

6/14/2013
Preliminary graphs of HIF-1a gene expression from the first 20 samples:





**6/5/2013**

**Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix)**

__RNA__ (20ul RXN)

// Total # rxns = 20 samples+ 20 duplicates + 3 NTCs + 1 error = 44 //
2x Sso Fast EvaGreen 10 x 44 = 440ul HIF Forward Primer 2-1 0.5 x 44 = 22ul HIF Reverse Primer 2-1 0.5 x 44 = 22ul DEPC H20 8 x 44 =352ul

//DEPC H20 =1ul// //cDNA template = 1ul//

// Total # rxns = 20 samples+ 20 duplicates + 3 NTCs + 1 error = 44 //
2x Sso Fast EvaGreen 10 x 44 = 440ul EF-1 Forward Primer 2 0.5 x 44 = 22ul EF-1 Reverse Primer 2 0.5 x 44 = 22ul DEPC H20 8 x 44 =352ul

//DEPC H20 =1ul// //cDNA template = 1ul//

Plate:

Results:


 * 6/7/2013 **
 * Reverse Transcription (Promega M-MLV Protocol) **
 * RT the 20 field samples to create cDNA**

A single reaction volume = **25uL**. The volume of RNA, primer(s) and M-MLV RT used are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.

1) //Calculate volume of RNA = 1ug of RNA://

2) //Transfer calculated volume(s) of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O//.
 * Sample || ng/ul || ug/ul || ul of RNA ||
 * 2 || 141.8 || 0.14 || 7.05 ||
 * 7 || 191.5 || 0.19 || 5.22 ||
 * 11 || 98.0 || 0.10 || 10.20 ||
 * 18 || 103.8 || 0.10 || 9.63 ||
 * 22 || 170.3 || 0.17 || 5.87 ||
 * 30 || 188.4 || 0.19 || 5.31 ||
 * 33 || 106.7 || 0.11 || 9.37 ||
 * 40 || 109.9 || 0.11 || 9.10 ||
 * 41 || 416.3 || 0.42 || 2.40 ||
 * 47 || 158.4 || 0.16 || 6.31 ||
 * 55 || 148.9 || 0.15 || 6.72 ||
 * 56 || 215.1 || 0.22 || 4.65 ||
 * 64 || 201.7 || 0.20 || 4.96 ||
 * 69 || 163.4 || 0.16 || 6.12 ||
 * 72 || 230.9 || 0.23 || 4.33 ||
 * 77 || 108.0 || 0.11 || 9.26 ||
 * 85 || 184.9 || 0.18 || 5.41 ||
 * 87 || 168.1 || 0.17 || 5.95 ||
 * 91 || 122.7 || 0.12 || 8.15 ||
 * 99 || 337.4 || 0.34 || 2.96 ||

3) //Add appropriate amount of primer to sample. Use 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT in this example). Total volume (RNA + primers) should equal 18.25uL//. 17.75ulRNA + 0.5ul Promega oligo dT = 18.25ul
 * Sample || ul of RNA || DEPC 0.1% ||
 * 2 || 7.05 || 10.7 ||
 * 7 || 5.22 || 12.5 ||
 * 11 || 10.20 || 7.5 ||
 * 18 || 9.63 || 8.1 ||
 * 22 || 5.87 || 11.9 ||
 * 30 || 5.31 || 12.4 ||
 * 33 || 9.37 || 8.4 ||
 * 40 || 9.10 || 8.7 ||
 * 41 || 2.40 || 15.3 ||
 * 47 || 6.31 || 11.4 ||
 * 55 || 6.72 || 11.0 ||
 * 56 || 4.65 || 13.1 ||
 * 64 || 4.96 || 12.8 ||
 * 69 || 6.12 || 11.6 ||
 * 72 || 4.33 || 13.4 ||
 * 77 || 9.26 || 8.5 ||
 * 85 || 5.41 || 12.3 ||
 * 87 || 5.95 || 11.8 ||
 * 91 || 8.15 || 9.6 ||
 * 99 || 2.96 || 14.8 ||

4) // Heat samples at 70C for 5 min in thermocycler. // 5) //Placed samples on ice IMMEDIATELY.//

6) //Made __**Master Mix** (21 RXNs)__//

__PER RXN__ 5 uL 5x Buffer (M-MLV RT Buffer) x 21 = 105ul 1.25 uL 2.5mM dNTPs x 21 = 26.3ul 0.5 uL M-MLV RT **per ug of RNA** x 21 **=** 10.5ul

//7) Mix well.// //8) Added 6.75uL of master mix to each reaction.// //9) Mix well, but do not vortex (invert)// //10) Spot spin.// //11) Incubate @ 42C for 1hr in thermalcycler for oligo dT primers OR @ 37C for random primers.// //12) Heat inactivate @ 95C for 3 min.// //13) Spot spin.// //14)Stored @ -20C.// = = =6/5/2013= **Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix)**

qPCR to get test 'clean' RNA samples compared to concentrated gDNA (4/10/2013):

__RNA__ (20ul RXN) __Master Mix:__ //2x Sso Fast EvaGreen 10 x 38 = 380ul// //Forward Primer 2 0.5 x 38 = 19ul// //Reverse Primer 2 0.5 x 38 = 19ul// //DEPC H20 8.5 x 38 =323ul//

//DEPC H20 = 0.5ul// //gDNA template = 0.5ul//

//Total # rxns = 32sampls + 3 NTCs + 2 gDNA + 1 error = 38//

Results: NTCs clean, no amplification of HIF for RNA samples. Proceed to reverse transcription of RNA.

=5/29/2013=

**Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix)** qPCR to get clean NTC compared to concentrated gDNA (4/10/2013):

__RNA__ (20ul RXN) __Master Mix:__ //2x Sso Fast EvaGreen 10 x 7 = 70ul// //Forward Primer 2 0.5 x 7 = 3.5ul// //Reverse Primer 2 0.5 x 7 = 3.5ul// //DEPC H20 8.5 x 7 =59.5ul//

//DEPC H20 = 0.5ul// //gDNA template = 0.5ul//

//Total # rxns = 4 NTCs + 2 gDNA + 1 error = 7//

Changed: 1) Bleached station 2) Used 1/3 fresh cut plate 3) Invert Sso Fast 4) Used same pipet tip to distribute master-mix 5) Used different pipet tip to distribute gDNA and NTC replicates 6) Wiped down top of plate (kim wipe) after placing in Bio-Rad

Results:

All NTCs clean!!!! - I will now run qPCR on a subsample of my RNA (5 out of 20 samples).

=4/11/2013=

**Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix)** qPCR on DNased RNA and newly concentrated gDNA (4/10/2013):

__RNA__ (20ul RXN) __Master Mix:__ //2x Sso Fast EvaGreen 10 x 8 = 80ul// //Forward Primer 2 0.5 x 8 = 4ul// //Reverse Primer 2 0.5 x 8 = 4ul// //H20 8.5 x 8 = 68ul//

//RNA template = 0.5ul// //gDNA template = 0.5ul//

Results: (for full results)

One replicate NTC (//dark blue//) and one replicate sample (#72; //aqua//) c ontaminated...otherwise, the remaining controls and samples look good.

=4/10/2013=

**Ethanol precipitate of gDNA in order to** concentrate
Made 25ml solution of 3M sodium acetate //Xg * 1mol/82.03g * 1/25ml * 1000ml/L = 3M// //Xg = 6.15g of sodium acetate//

1) 100ul of 'old' gDNA (//see 12/5/2013 for DNA extraction//) 2) Add 0.1 volumes (10ul) of 3M sodium acetate 3) incubate at -20C for at least 30min 4) pellet DNA at 16,000g for 15mins at 4C 5) wash pellet with 1ml of 70% ethanol 6) pellet DNA at 16,000g for 15 mins at 4C 7) discard supernatant 8) resuspend pellet (gently) in desired volume (50ul) of water/buffer

Nanospec
 * **gDNA ng/ul** || **260/280** || **260/230** ||
 * 188.8 || 1.78 || 1.38 ||

**Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix)** qPCR on DNased RNA and newly concentrated gDNA (above):

__RNA__ (20ul RXN) __Master Mix:__ //2x Sso Fast EvaGreen 10 x 16 = 160ul// //Forward Primer 2 0.5 x 16 = 8ul// //Reverse Primer 2 0.5 x 16 = 8ul// //H20 8.5 x 16 = 136ul//

//RNA template = 0.5ul// // gDNA template = 0.5ul //

Results:

Relative to the gDNA, the samples look relatively good...but, yet again, only one of my NTC controls is clean.

=12/20/2012=

Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix)
qPCR on DNased RNA and new EF-1a primers:

__RNA__ (20ul RXN) __Master Mix:__ //2x Sso Fast EvaGreen 10 x 46 = 460ul// //Forward Primer 2 0.5 x 46 = 23ul// //Reverse Primer 2 0.5 x 46 = 23ul// //H20 8.5 x 46 = 391ul//

//RNA template = 0.5ul//

__EF-1a Primer Pair 1 & Primer Pair 2__ __Two Seperate Master Mixes:__ //2x Sso Fast EvaGreen 10 x 7= 70ul// //Forward Primer 2 0.5 x 7= 3.5ul// //Reverse Primer 2 0.5 x 7= 3.5ul// //H20 8 x 7= 56ul//

//cDNA-1 template = 1ul//

RESULTS: One NTC was clean. Although most of the samples showed none or reduced amplification, some samples exhibited that early florescence...

Both primers appear to work, however EF-2 had much more amplification.

=12/19/2012=

**TURBO DNA-free Protocol**
1. Each RNA sample was diluted to equal 10ug of RNA for 50ul rxn in a **0.5mL tube**:


 * Sample || ug/ul || Dilution for 10ug of RNA || 0.1% DEPC ||
 * 2 || 1.18 || 8.50 || 41.5 ||
 * 7 || 1.14 || 8.78 || 41.2 ||
 * 11 || 1.27 || 7.89 || 42.1 ||
 * 18 || 0.94 || 10.68 || 39.3 ||
 * 22 || 0.78 || 12.77 || 37.2 ||
 * 30 || 0.78 || 12.78 || 37.2 ||
 * 33 || 1.19 || 8.41 || 41.6 ||
 * 40 || 0.74 || 13.53 || 36.5 ||
 * 41 || 1.26 || 7.94 || 42.1 ||
 * 47 || 1.37 || 7.31 || 42.7 ||
 * 55 || 0.85 || 11.81 || 38.2 ||
 * 56 || 1.39 || 7.21 || 42.8 ||
 * 64 || 0.26 || 38.25 || 11.8 ||
 * 69 || 0.41 || 24.39 || 25.6 ||
 * 72 || 1.47 || 6.80 || 43.2 ||
 * 77 || 2.44 || 4.09 || 45.9 ||
 * 87 || 1.07 || 9.37 || 40.6 ||
 * 85 || 1.75 || 5.72 || 44.3 ||
 * 91 || 1.53 || 6.54 || 43.5 ||
 * 99 || 1.49 || 6.70 || 43.3 ||

2. 5ul of the TURBO DNase buffer to each sample 3. 0.5ul of TURBO DNase was added to each sample 4. Sample were incubated for 30min @ 37C 5. Remaining 0.5ul of TURBO DNase was added to each sample (for a total of 1ul of DNase enzyme) 6. DNase Inactivation Reagent was resuspended (flicked) 7. 5ul of the Inactivation Reagent was added to each sample 8. Samples incubated for ~2min @ RT, each mixed/flicked an additional time to resuspend the reagent 9. Samples centrifuge at 10,000 x g for 1.5min 10. Supernatent was carefully pipetted to new (labeled) 0.5mL tubes.

"Clean" RNA was then spec'ed in the Nano-Drop:
 * Sample || ng/ul || 260/280 || 260/230 ||
 * 2 || 141.8 || 1.96 || 1.09 ||
 * 7 || 191.5 || 1.97 || 1.00 ||
 * 11 || 98.0 || 1.95 || 1.29 ||
 * 18 || 103.8 || 1.97 || 0.77 ||
 * 22 || 170.3 || 1.97 || 1.29 ||
 * 30 || 188.4 || 1.95 || 0.78 ||
 * 33 || 106.7 || 1.93 || 1.29 ||
 * 40 || 109.9 || 1.98 || 1.10 ||
 * 41 || 416.3 || 1.95 || 1.56 ||
 * 47 || 158.4 || 1.97 || 1.51 ||
 * 55 || 148.9 || 1.97 || 1.31 ||
 * 56 || 215.1 || 1.99 || 1.45 ||
 * 64 || 201.7 || 1.98 || 1.52 ||
 * 69 || 163.4 || 2.00 || 1.46 ||
 * 72 || 230.9 || 1.99 || 1.54 ||
 * 77 || 108.0 || 1.90 || 1.11 ||
 * 85 || 184.9 || 1.98 || 1.28 ||
 * 87 || 168.1 || 1.97 || 1.18 ||
 * 91 || 122.7 || 1.94 || 1.29 ||
 * 99 || 337.4 || 1.97 || 1.49 ||

=12/11/2012= The ConsensusfromContig6914 (Q92005) Elongation factor 1-alpha for Pacific herring (Herring Hepatic Transcriptome) was BLASTed in NCBI to evaluate the most conserved region. Primer BLAST then was used on the contig sequence and two primers were selected and order from IDT:



=12/6/2012=
 * Primer pair 1 **
 * || **Sequence (5'->3') ** || **Template strand ** || **Length ** || **Start ** || **Stop ** || **Tm ** || **GC% ** || **Self complementarity ** || **Self 3' complementarity ** ||
 * **Forward primer ** || CTCCGCATTTGTAGATGAGA || Plus || 20 || 2414 || 2433 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">54.98 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">45.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">4.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2.00 ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Reverse primer ** || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">CTTAAGCAATCATGGGCAAG || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Minus || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">20 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2521 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2502 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">55.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">45.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">6.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2.00 ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Product length ** |||||||||||||||||| <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">108 ||
 * <span style="color: #336699; font-family: 'Times New Roman',serif; font-size: 8pt;">Primer pair 2 **
 * || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Sequence (5'->3') ** || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Template strand ** || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Length ** || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Start ** || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Stop ** || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Tm ** || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">GC% ** || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Self complementarity ** || **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Self 3' complementarity ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Forward primer ** || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">AGAGCAATGTCAATGGTGAT || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Plus || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">20 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2281 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2300 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">55.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">40.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">5.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2.00 ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Reverse primer ** || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">TCTCATCTACAAATGCGGAG || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Minus || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">20 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2433 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2414 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">54.98 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">45.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">4.00 || <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">2.00 ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 8pt;">Product length ** |||||||||||||||||| <span style="font-family: 'Times New Roman',serif; font-size: 8pt;">153 ||

Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix)
"Test" qPCR for primers and corrected cDNA and gDNA:

__No. of RXNs__ 2 cDNA-1 2 cDNA-2 2 gDNA 2 NTCs 1 pipette error = 9 RXNs

__Master Mix:__ //2x Sso Fast EvaGreen 10 x 9 = 90ul// //Forward Primer 2 0.5 x 9 = 4.5ul// //Reverse Primer 2 0.5 x 9 = 4.5ul// //H20 8 x 9 = 72ul//

//cDNA template = 1ul (**instead of 2ul)//

Order (//place in columns 6 & 7//): S --- cDNA-1 cDNA-1 cDNA-2 cDNA-2 gDNA gDNA

N --- NTC-1 NTC-2



RESULTS: Clean amplification (i.e., no early florescence), duplicates almost identical, a singular peak for all samples. However, one of the two NTCs was contaminated. I will now proceed to DNasing my 'real' RNA samples and run a qPCR on that RNA to assure purity of samples.

=12/5/2012=

Nano-drop Spec. results on Robert's Lab Herring RNA:


 * **Sample** || **ng/ul** || **260/280** || **260/230** ||
 * RNA-1 || 771.3 || 2.07 || 1.66 ||
 * RNA-2 || 1786.8 || 2.00 || 2.02 ||


 * Reverse Transcription (Promega M-MLV Protocol) **

A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.

1) //Calculate volume of RNA = 1ug of RNA:// RNA-1 = 0.7713 ug/ul * Xul of RNA = 1ug of RNA RNA-2 = 1.79 ug/ul * Xul of RNA = 1ug of RNA

2) //Transfer calculated volume(s) of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O//. V of RNA-1 = 1.3ul --> 1.3ul + 16.5ul DEPC 0.1% = 17.75ul V of RNA-2 = 0.56ul --> 0.56ul + 17.19ul DEPC 0.1% = 17.75ul

3) //Add appropriate amount of primer to sample. Use 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT in this example). Total volume (RNA + primers) should equal 18.25uL//. 17.75ulRNA-1 + 0.5ul Promega oligo dT = 18.25ul 17.75ulRNA-2 + 0.5ul Promega oligo dT = 18.25ul

4) //Heat samples at 70C for 5 min in thermocycler.// 5) //Placed samples on ice IMMEDIATELY.//

6) //Made __**Master Mix** (3 RXNs)__//

__PER RXN__ 5 uL 5x Buffer (M-MLV RT Buffer) x 3 = 15ul 1.25 uL 10mM dNTPs x 3 = 3.75ul 0.5 uL M-MLV RT **per ug of RNA =**1.5ul

//7) Mix well.// //8) Added 6.75uL of master mix to each reaction.// //9) Mix well, but do not vortex (invert)// //10) Spot spin.// //11) Incubate @ 42C for 1hr in thermalcycler for oligo dT primers OR @ 37C for random primers.// //12) Heat inactivate @ 95C for 3 min.// //13) Spot spin.// //14)Stored @ -20C.//

**DNA Extraction with DNazol**
1) With a sterile pestile, homogenized tissue sample (Herring liver: field sample #7) in 500ul of DNazol in a 1.5ml sterile micorfuge tube. After homogenized, an additional 500ul of DZanol was added and mixed well (flicked and slowly inverted) 2) Sampled incubated for 5min @ room temperature (RT) 3) Sample spun at 10,000 x g (RT) for 10min 4) Transfer supernatant to a new, labeled 1.5 micofuge tube 5) Added 500ul of 100% ethanol (EtOH) to supernatant 6) Mixed by inverting (slowly/gently) 5-8 times 7) Stored sample at RT for 1min 8) DNA was a cloudy precipitate. Degraded DNA & small quantities of DNA (<15ug) do not spool onto a pipette tip. So, the DNA precipitate was sedimentized by centrifugation @ 5,000g for 5min at 4C (NOTE: White DNA precipitate clung to side of tube after centrifuge). 9) The liquid was carefully discarded 10) The sample sat for 1min @ RT then the rest of the lysate (liquid that is not the DNA) was removed 11) DNA was washed with 1000ul of 75% EtOH: EtOH pipetted into the DNA tube, carefully inverted 6 times, and let sit for 1 min. Ethanol was then removed and this step was repeated. 12) Remaining EtOH at the bottom of the tube after the 2nd wash was removed with a small pipette 13) 300ul of 0.1% DEPC water was then added to the DNA and slowly pipetted up and down multiple time to dissolve DNA into solution 14) DNA sample was then quantified using Nanodrop (see result below):


 * **Sample** || **ng/ul** || **260/280** || **260/230** ||
 * DNA-7 || 103.4 || 1.64 || 0.79 ||

=11/27/2012=

__ Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix) [Cost per rxn ~$0.42] __
Single reaction (20uL) set up is listed below with the FRESH primers:

__Master Mix:__ //2x Sso Fast EvaGreen 10 x 4 = 40ul// //Forward Primer 2 0.5 x 4 = 2ul// //Reverse Primer 2 0.5 x 4 = 2ul// //H20 7 x 4 = 70ul//

//cDNA template = 2ul//

Order: A --- P2 NTC NTC



Amplification curve still fluorescing too soon. Sam is going to take a stab at it to pin point the problem - possibly 'wonky' cDNA? Good news, the control samples are clean!

__**Conventional PCR was preformed on the Pacific herring (liver) cDNA:**__

__//MASTER MIX CALCULATIONS://__

//12.5ul 2xApex Red *24 = 300ul// //0.5ul Forward primer *24= 12ul// //0.5ul Reverse primer// //*24 = 12ul// //9.5ul PCR H20 *24 = 228ul//

//Above calculations used for primer pair #2//

//1 cycle:// 95C - 10mins
 * Cycling parameters:**

//39 cyles of:// 95C - 15s 72C - 30sec
 * 62C** - 15s

//1 cycle:// 72C - 8min 4C - Inf.

Ran one medium agarose gel for primer pair 2: 100ml 1x TAE 1.90 Agarose 10 EtBr

//Loaded 5ul of Hyperladder II// //8ul of sample into each well (primer pair & NTCs)//

//The thermocycler malfunctioned in the beginning (over heated my samples) which may have degraded the samples and thus the results. The NTCs were clean (not shown).//

=11/16/2012= Nano-drop Results from RNA (20 samples). Clean RNA: **260/280** range should be **1.8-2.0** and **260/230** should range between **1.5-2.0**.


 * Sample # || ng/ul || 260/280 || 260/230 ||
 * 2 || 1176.51 || 2.01 || 1.43 ||
 * 7 || 1139.20 || 2.00 || 1.34 ||
 * 11 || 1267.65 || 1.99 || 1.96 ||
 * 18 || 936.53 || 1.98 || 1.86 ||
 * 22 || 782.78 || 1.99 || 1.74 ||
 * 30 || 782.51 || 1.99 || 0.97 ||
 * 33 || 1188.60 || 2.01 || 1.95 ||
 * 40 || 739.16 || 1.98 || 1.74 ||
 * 41 || 1259.86 || 2.01 || 1.04 ||
 * 47 || 1367.16 || 2.02 || 2.12 ||
 * 55 || 846.43 || 1.99 || 1.80 ||
 * 56 || 1386.20 || 2.02 || 1.19 ||
 * 64 || 261.45 || 1.84 || 2.01 ||
 * 69 || 409.99 || 1.86 || 1.99 ||
 * 72 || 1471.31 || 2.02 || 1.91 ||
 * 77 || 2442.94 || 1.96 || 1.69 ||
 * 87 || 1067.66 || 2.02 || 1.08 ||
 * 85 || 1749.34 || 2.02 || 1.58 ||
 * 91 || 1529.40 || 2.02 || 1.98 ||
 * 99 || 1493.62 || 2.01 || 1.57 ||
 * //**Mean**// || **//1164.92//** || //**1.99**// || //**1.65**// ||
 * //**SD**// || //**483.58**// || //**0.05**// || //**0.36**// ||

__** Reverse Transcription (RT) of the 20 Pacific herring liver RNA samples (above): **__ 1. Once our stock RNA thawed, inverted tube to mix sample. 2. Labeled 0.5ml PCR tube with "Sample # cDNA". 3. Pipetted 5µl of our stock RNA into the PCR tube 4. Added 1µl of oligo dT 5. Added 4µl of nuclease free H2O (i.e., DEPC) 6. In a thermocycler, we incubated the mix at 70C for 5min 7. After incubation, we place the mix on ice for 2min 8. Spun the sample down 9. Added 5µl of M-MLV 5x Reaction buffer 10. Added 5µl of dNTPs (2.5uM) 11. Added 1µl of M-MLV Reverse Transcriptase (RT) 12. Added 4µl more of M-MLV nuclease free H2O 13. Vortexed the mix for several seconds 14. Spun it down 15. Placed back in the thermocycler, the mix was incubated for 60min at 42C, then heat inactivate at 94C for 3min. 16. Mix spun down and kept on ice (ultimately stored @ -20C).

Waiting for fresh (uncontaminated) primers to proceed...

=11/15/2012= Finished extracting the RNA from homogenized samples (10/31/2012)

=11/13/2012= //***qPCR run on 11/9/2012 resulted in odd results. So, to ensure clean samples and NTCs a new batch of primer working stock (10mM) and PCR H2O were used. In addition, the plate was placed in the center of the thermocycler.***//

Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix) [Cost per rxn ~$0.42]
Single reaction (20uL) set up is listed below:

__Master Mix:__ //2x Sso Fast EvaGreen 10 x 5 = 50ul// //Forward Primer 2 0.5 x 5 = 2.5ul// //Reverse Primer 2 0.5 x 5 = 2.5ul// //H20 7 x 5 = 35ul//

//2ul Template//

Order: A --- P2 P2 NTC NTC

RESULTS:

Problems: 1) Amplification of product in NTCs -- SOLUTION: Ordered new Primer Pair 2 from IDT
 * <span style="color: #336699; font-family: Verdana,sans-serif; font-size: 10.5pt;">Primer pair 2 **
 * || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Sequence (5'->3') ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Template strand ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Length ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Start ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Stop ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Tm ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">GC% ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Self complementarity ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Self 3' complementarity ** ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Forward primer ** || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">GTTGAGCAGCTTCCTCATGC || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Plus || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">20 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2498 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2517 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">53.85 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">55.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">6.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2.00 ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Reverse primer ** || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">GGAGTCGGAGGTGTTCTACG || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Minus || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">20 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2626 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2607 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">53.85 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">60.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">3.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2.00 ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Product length ** |||||||||||||||||| <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">129 ||

2) Irregular amplification for rxns with template -- Possible reasons:

**Sigmoidal** **amplification curves** If your amplification curves look sigmoidal, it is likely you have one of these problems:
 * <span style="color: #1279c6; font-family: Verdana,sans-serif; font-size: 8pt;">[|The baseline setting in your instrument’s data analysis software may be too low]
 * <span style="color: #1279c6; font-family: Verdana,sans-serif; font-size: 8pt;">[|You may have a high level of fluorescent noise during the early cycles of PCR]

=11/9/2012=

Quantitative PCR (qPCR)/Real-time PCR (2x Sso Fast EvaGreen Supermix) [Cost per rxn ~$0.42]
Single reaction (20uL) set up is listed below. Be sure to make a master mix volume that will accommodate the following: all of your samples, two water (no template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. Distribute appropriate amount of master mix (volume of master mix + template = 20uL) to white PCR plate. Add template. Cap with optical PCR caps. Spin plate for 1min @ 3000g. Put in thermalcycler.


 * **Reaction_Components** || **Volume** || **Final Concentration** ||
 * 2x Sso Fast EvaGreen || 10 || 1x ||
 * Forward Primer (10uM) || 0.5 || 0.2uM ||
 * Reverse Primer (10uM) || 0.5 || 0.2uM ||
 * Template || Up to 5uL ||  ||
 * H2O (PCR grade) || variable || Use to bring reaction volume up to 20uL ||

__Master Mix:__ //2x Sso Fast EvaGreen 10 x 5 = 50ul// //Forward Primer 2 0.5 x 5 = 2.5ul// //Reverse Primer 2 0.5 x 5 = 2.5ul// //H20 7 x 5 = 35ul//

RESULTS: Funky....redo.

=11/2/2012= __**Conventional PCR was preformed on the Pacific herring (liver) cDNA:**__

__//MASTER MIX CALCULATIONS://__

//12.5ul 2xApex Red *4 = 50ul// //0.5ul Forward primer *4= 2ul// //0.5ul Reverse primer// //*4 = 2ul// //9.5ul PCR H20 *4 = 38ul//

//12.5ul 2xApex Red *4 = 50ul 75% volume of primers to reduce primer-dimers// //0.375ul Forward primer *4= 1.5ul// //0.375ul Reverse primer// //*4 = 1.5ul// //9.75ul PCR H20 *4 = 39ul//

//Above calculations used for each primer pair #2//

//1 cycle:// 95C - 10mins
 * Cycling parameters:**

//39 cyles of:// 95C - 15s 72C - 30sec
 * 62C** - 15s

//1 cycle:// 72C - 8min 4C - Inf.

Ran one small agarose gel for primer pair 2:

//Loaded 5ul of Hyperladder II// //12ul of sample into each well (primer pair & NTCs)//



__Results__//: Controls clean, primer-dimers reduced, and product maintained (although not as tight a band as I would like...but I'll take what I can get!) using full volume (0.5ul) of primer. Next step use qPCR to determine melt and amplification quantities.//

=10/31/2012= __**Conventional PCR was preformed on the Pacific herring (liver) cDNA:**__ //Conventional PCR (2x Apex Red)//** Single reaction (25uL) set up is listed below. Be sure to make a master mix volume that will accommodate all of your samples, two water (non-template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. Distribute appropriate amount of master mix (volume of master mix + template = 25uL) to PCR tubes or PCR plate. Make sure all tubes/caps are tightly closed. Put in thermalcycler. __//MASTER MIX CALCULATIONS: **NOTE: Primer volume was halved (0.5ul to 0.25ul) to reduce primer-dimers**//__ //12.5ul 2xApex Red *4 = 50ul// //0.25ul Forward primer *4= 1ul// //0.25ul Reverse primer// //*4 = 1ul// //10ul PCR H20 *4 = 40ul// //Above calculations used for each primer pair #2//
 * **Reaction_Components** || **Volume** || **Final Concentration** ||
 * 2x Apex Red || 12.5 || 1x ||
 * Forward Primer (10uM) || 0.25 || 0.2uM ||
 * Reverse Primer (10uM) || 0.25 || 0.2uM ||
 * Template || Up to 5uL ||  ||
 * H2O (PCR grade) || variable || Use to bring reaction volume up to 25uL ||

//1 cycle:// 95C - 10mins
 * Cycling parameters:**

//39 cyles of:// 95C - 15s 72C - 30sec
 * 62C** - 15s

//1 cycle:// 72C - 8min 4C - Inf.

Ran one small agarose gel for primer pair 2 (//see// 5/30/2012). //TAE 75ml// //Agarose 1g// //EtBr 5ul//

//Loaded 5ul of Hyperladder II// //12ul of sample into each well (primer pair & NTCs)//




 * Results:** The primer-dimers were reduced in the NTC, but now there is a "smear" for the sample rxn with no clear band.

1) ~0.002-0.003g of liver tissue (n=20) was homogenized with a sterile pestle in 500ul of TriReagent in a 1.5ml microfuge tube 2) Shortly vortex 3) Additional 500ul of TriReagent was then added to the test tube 4) Vortexed for 15s 5) Homogenized tissues were incubated at RT for 5min 6) Stored at -80C
 * __On Herring Field Samples (see 10/23/2012 for samples processed)__**
 * RNA Extraction ****

=10/29/2012=

Ran one medium agarose gel for primer pair 2 (//see// 5/30/2012). //TAE 125ml// //Agarose 1.72g// //EtBr 12ul//

//Loaded 5ul of Hyperladder II// //12ul of sample into each well (primer pair & NTCs)//



__**Results:**__ Temperatures greater than or equal to 63C should be avoided. However, the primer-dimers are still a nightmare.

=10/25/2012=

__**Conventional PCR was preformed on the Pacific herring (liver) cDNA:**__ //Conventional PCR (2x Apex Red)//** Single reaction (25uL) set up is listed below. Be sure to make a master mix volume that will accommodate all of your samples, two water (non-template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. Distribute appropriate amount of master mix (volume of master mix + template = 25uL) to PCR tubes or PCR plate. Make sure all tubes/caps are tightly closed. Put in thermalcycler. __//MASTER MIX CALCULATIONS://__ //12.5ul 2xApex Red *25 = 312.5ul// //0.5ul Forward primer *25= 12.5ul// //0.5ul Reverse primer// //*25 = 12.5ul// //9.5ul PCR H20 *25 = 237.5ul// //**Above calculations used for each primer pair #2**//
 * **Reaction_Components** || **Volume** || **Final Concentration** ||
 * 2x Apex Red || 12.5 || 1x ||
 * Forward Primer (10uM) || 0.5 || 0.2uM ||
 * Reverse Primer (10uM) || 0.5 || 0.2uM ||
 * Template || Up to 5uL ||  ||
 * H2O (PCR grade) || variable || Use to bring reaction volume up to 25uL ||

Cycling parameters: //1 cycle:// 95C - 10mins

//39 cyles of:// 95C - 30s 60-65C - 30s **//NOTE: PCR was conducted in the Real Time PCR Detector to perform a temp gradient (60C-65C) of annealing temperatures//** 72C - 90sec

//1 cycle:// 72C - 3min 4C - Inf.

Temp gradient:
 * || **60C** || **60.2C** || **60.6C** || **61.2C** || **62C** || **63C** || **64.2C** || **65.2C** ||
 * || **1** || **2** || **3** || **4** || **5** || **6** || **7** || **8** ||
 * **A** || P2-1 || P2-2 || P2-3 || P2-4 || P2-5 || P2-6 || P2-7 || P2-8 ||
 * **B** || N1-1 || N1-2 || N1-3 || N1-4 || N1-5 || N1-6 || N1-7 || N1-8 ||
 * **C** || N2-1 || N2-2 || N2-3 || N2-4 || N2-5 || N2-6 || N2-7 || N2-8 ||

=10/24/2012=

__** Reverse Transcription (RT) of Pacific herring liver RNA provided by the Robert's Lab: **__ 1. Once our stock RNA thawed, inverted tube to mix sample. 2. Labeled 0.5ml PCR tube with "HIF cDNA". 3. Pipetted 5µl of our stock RNA into the PCR tube 4. Added 1µl of oligo dT 5. Added 4µl of nuclease free H2O (i.e., DEPC) 6. In a thermocycler, we incubated the mix at 70C for 5min 7. After incubation, we place the mix on ice for 2min 8. Spun the sample down 9. Added 5µl of M-MLV 5x Reaction buffer 10. Added 5µl of dNTPs (2.5uM) 11. Added 1µl of M-MLV Reverse Transcriptase (RT) 12. Added 4µl more of M-MLV nuclease free H2O 13. Vortexed the mix for several seconds 14. Spun it down 15. Placed back in the thermocycler, the mix was incubated for 60min at 42C, then heat inactivate at 94C for 3min. 16. Mix spun down and kept on ice (ultimately stored @ -20C).

=10/23/2012=

All 100 liver tissue samples have been collected from the field.



20 samples will be processed at a time; the first 20 (in green) have been randomly selected to include five samples from each collection month and station.



=8/23/2012= Ran one small agarose gel for primer pair 2 (//see// 5/30/2012). //(gel from 8/21/12)//

//Loaded 5ul of Hyperladder II// //20ul of samples (primer pair & NTCs)//


 * Results:**



Got rid of the primer-dimers, but at the expense on the PCR product. Need to test smaller annealing thermoregimes with the 60-65C range.

=8/21/2012= __Conventional PCR was preformed on the Pacific herring (liver) cDNA (see 6/20/12):__ Single reaction (25uL) set up is listed below. Be sure to make a master mix volume that will accommodate all of your samples, two water (non-template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. Distribute appropriate amount of master mix (volume of master mix + template = 25uL) to PCR tubes. Make sure all tubes/caps are tightly closed. Put in thermalcycler. __//MASTER MIX CALCULATIONS://__ //12.5ul 2xApex Red *4 = 50ul// //0.5ul Forward primer *4 = 2ul// //0.5ul Reverse primer// //*4 = 2ul// //9.5ul PCR H20 *4 = 38ul// //**Above calculations used for primer pair 2**//
 * //Conventional PCR (2x Apex Red)//**
 * **Reaction_Components** || **Volume** || **Final Concentration** ||
 * 2x Apex Red || 12.5 || 1x ||
 * Forward Primer (10uM) || 0.5 || 0.2uM ||
 * Reverse Primer (10uM) || 0.5 || 0.2uM ||
 * Template || Up to 5uL ||  ||
 * H2O (PCR grade) || variable || Use to bring reaction volume up to 25uL ||

Cycling parameters: //1 cycle:// 95C - 10mins

//39 cyles of:// 95C - 15s 72C - 30sec
 * 65C** - 15s **//NOTE: Annealing temperature was increased from 60C to 65C to remove primer-dimers//**

//1 cycle:// 72C - 8min 4C - Inf.

Also prepared small agarose gel (16 wells) 75ml of 1XTAE 1g agarose 6ul of EtBr

=8/15/12= __Conventional PCR was preformed on the Pacific herring (liver) cDNA (see 6/20/12):__ Single reaction (25uL) set up is listed below. Be sure to make a master mix volume that will accommodate all of your samples, two water (non-template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. Distribute appropriate amount of master mix (volume of master mix + template = 25uL) to PCR tubes. Make sure all tubes/caps are tightly closed. Put in thermalcycler. __//MASTER MIX CALCULATIONS://__ //12.5ul 2xApex Red *4 = 50ul// //0.5ul Forward primer *4 = 2ul// //0.5ul Reverse primer// //*4 = 2ul// //9.5ul PCR H20 *4 = 38ul// //**Above calculations used for all three primer pairs**//
 * //Conventional PCR (2x Apex Red)//**
 * **Reaction_Components** || **Volume** || **Final Concentration** ||
 * 2x Apex Red || 12.5 || 1x ||
 * Forward Primer (10uM) || 0.5 || 0.2uM ||
 * Reverse Primer (10uM) || 0.5 || 0.2uM ||
 * Template || Up to 5uL ||  ||
 * H2O (PCR grade) || variable || Use to bring reaction volume up to 25uL ||

Cycling parameters: //1 cycle:// 95C - 10mins

//39 cyles of:// 95C - 15s 72C - 30sec
 * 60C** - 15s **//NOTE: Annealing temperature was increased from 55C to 60C to remove primer-dimers//**

//1 cycle:// 72C - 8min 4C - Inf.

Ran one small agarose gel for all primer pairs (//see// 5/30/2012). //(gel from 8/13/12)//

//Placed gel in 1X TAE box (with comb)// //Removed comb// //Loaded 5ul of Hyperladder II// //12ul of samples (primer pairs & NTCs)//


 * Results:**



Still have primer-dimers. Stumped. But primer 4 is totally out of the running!

= = =8/13/12=

Ran one small agarose gel for two of the primer pairs (//see// 5/30/2012). //75ml of 1x TAE// //1g of agrarose// //Microwaved ~3min, mix// //6ul of Ethedium Bromide (EtBr)// //Mix// //Pour into container// //Place well-comb// //Let sit for ~30min//

//Placed gel in 1X TAE box (with comb)// //Removed comb// //Loaded 7ul of Hyperladder II// //15ul of samples (primer pairs & NTCs)//


 * Results:**

Still have primer-dimers. Will try another round (8/15/2012) of cPCR with all three primers at an annealing temp of 60C (instead of 55C).

=8/03/12= __Conventional PCR was preformed on the Pacific herring (liver) cDNA (see 6/20/12):__ Single reaction (25uL) set up is listed below. Be sure to make a master mix volume that will accommodate all of your samples, two water (non-template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. Distribute appropriate amount of master mix (volume of master mix + template = 25uL) to PCR tubes. Make sure all tubes/caps are tightly closed. Put in thermalcycler. __//MASTER MIX CALCULATIONS://__ //12.5ul 2xApex Red *4 = 50ul// //0.5ul Forward primer *4 = 2ul// //0.5ul Reverse primer// //*4 = 2ul// //9.5ul PCR H20 *4 = 38ul// //**Above calculations used for each set of primer pairs (only using primer pair 1 and 2, no 4)**//
 * //Conventional PCR (2x Apex Red)//**
 * **Reaction_Components** || **Volume** || **Final Concentration** ||
 * 2x Apex Red || 12.5 || 1x ||
 * Forward Primer (10uM) || 0.5 || 0.2uM ||
 * Reverse Primer (10uM) || 0.5 || 0.2uM ||
 * Template || Up to 5uL ||  ||
 * H2O (PCR grade) || variable || Use to bring reaction volume up to 25uL ||

Cycling parameters: //1 cycle:// 95C - 10mins

//39 cyles of:// 95C - 15s 72C - 30sec
 * 55C** - 15s **//NOTE: Annealing temperature was increased from 50C to 55C to remove primer-dimers//**

//1 cycle:// 72C - 8min 4C - Inf.

Stored at 20C for running gel on Monday 8/13/2012

= = =6/27/2012*=

Ran one medium agarose gel for all three primer pairs (//see// **5/30/2012**). //150ml of 1x TAE// //2g of agrarose// //Microwaved ~3min, mix// //12ul of Ethedium Bromide (EtBr)// //Mix//

//Results://

Hyperladder II in the 1st well, 3 samples and 2 non-template controls (NTCs) for each primer pair in the remaining wells.

Ran gel for 50 min at 100v


 * Results:**
 * Primer 1: 116bp**
 * Primer 2: 129bp**
 * Primer 4: 138bp**

Primer 1 & 2 products were the correct size (between 100-200bp, closer to 100bp) and the NTCs were clean (although the first NTC 2 did have a very slight band). Primer 4 appears contaminated. = = =6/20/2012*=

__** Reverse Transcription (RT) of Pacific herring liver RNA provided by the Robert's Lab: **__ 1. Once our stock RNA thawed, inverted tube to mix sample. 2. Labeled 0.5ml PCR tube with "HIF cDNA". 3. Pipetted 5µl of our stock RNA into the PCR tube 4. Added 1µl of oligo dT 5. Added 4µl of nuclease free H2O (i.e., DEPC) 6. In a thermocycler, we incubated the mix at 70C for 5min 7. After incubation, we place the mix on ice for 2min 8. Spun the sample down 9. Added 5µl of M-MLV 5x Reaction buffer 10. Added 5µl of dNTPs (2.5uM) 11. Added 1µl of M-MLV Reverse Transcriptase (RT) 12. Added 4µl more of M-MLV nuclease free H2O 13. Vortexed the mix for several seconds 14. Spun it down 15. Placed back in the thermocycler, the mix was incubated for 60min at 42C, then heat inactivate at 94C for 3min. 16. Mix spun down and kept on ice (ultimately stored @ -20C).

__**Conventional PCR was preformed on the Pacific herring (liver) cDNA (from above):**__ //Conventional PCR (2x Apex Red)//** Single reaction (25uL) set up is listed below. Be sure to make a master mix volume that will accommodate all of your samples, two water (non-template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. Distribute appropriate amount of master mix (volume of master mix + template = 25uL) to PCR tubes or PCR plate. Make sure all tubes/caps are tightly closed. Put in thermalcycler. __//MASTER MIX CALCULATIONS://__ //12.5ul 2xApex Red *4 = 50ul// //0.5ul Forward primer *4 = 2ul// //0.5ul Reverse primer// //*4 = 2ul// //9.5ul PCR H20 *4 = 38ul// //**Above calculations used for each set of primer pairs (i.e., 3 x MM)**//
 * **Reaction_Components** || **Volume** || **Final Concentration** ||
 * 2x Apex Red || 12.5 || 1x ||
 * Forward Primer (10uM) || 0.5 || 0.2uM ||
 * Reverse Primer (10uM) || 0.5 || 0.2uM ||
 * Template || Up to 5uL ||  ||
 * H2O (PCR grade) || variable || Use to bring reaction volume up to 25uL ||

Cycling parameters:** 95C - 10mins 40 cyles of: 95C - 15s 50C - 15s 72C - 10s - 2mins (dependent on amplicon size; ~1000kb/min)

Stored at 20C for running gel on Friday 6/22/2012

=6/11/ - 6/15/2012*=

Collected liver tissue samples from four sites in Hood Canal, WA; 5 specimen from each site (20 total herring). On board, liver tissue samples (~1 mg) were collected and preserved in 5ml of RNAlater. Back at the lab, samples were stored at -20C.

=5/30/2012*=


 * Ordered three primer pairs (IDT) for Pacific herring (//Clupea pallasii//) HIF-1a mRNA analysis:**


 * <span style="color: #336699; font-family: Verdana,sans-serif; font-size: 10.5pt;">Primer pair 1 **
 * || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Sequence (5'->3') ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Template strand ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Length ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Start ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Stop ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Tm ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">GC% ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Self complementarity ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Self 3' complementarity ** ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Forward primer ** || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">AGTGGAGCACCTTCCATGTG || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Plus || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">20 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2140 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2159 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">54.09 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">55.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">4.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">3.00 ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Reverse primer ** || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">CAAGAAGGGCAAGGAGCAGA || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Minus || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">20 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2255 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2236 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">54.09 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">55.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">0.00 ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Product length ** |||||||||||||||||| <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">116 ||


 * <span style="color: #336699; font-family: Verdana,sans-serif; font-size: 10.5pt;">Primer pair 2 **
 * || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Sequence (5'->3') ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Template strand ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Length ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Start ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Stop ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Tm ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">GC% ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Self complementarity ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Self 3' complementarity ** ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Forward primer ** || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">GTTGAGCAGCTTCCTCATGC || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Plus || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">20 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2498 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2517 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">53.85 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">55.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">6.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2.00 ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Reverse primer ** || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">GGAGTCGGAGGTGTTCTACG || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Minus || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">20 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2626 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2607 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">53.85 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">60.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">3.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2.00 ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Product length ** |||||||||||||||||| <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">129 ||


 * <span style="color: #336699; font-family: Verdana,sans-serif; font-size: 10.5pt;">Primer pair 4 **
 * || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Sequence (5'->3') ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Template strand ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Length ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Start ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Stop ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Tm ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">GC% ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Self complementarity ** || **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Self 3' complementarity ** ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Forward primer ** || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">CACCTTCCATGTGGAGGACT || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Plus || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">20 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2147 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2166 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">53.09 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">55.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">6.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2.00 ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Reverse primer ** || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">AGGGAGATGTTGGTCCACAG || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Minus || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">20 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2284 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">2265 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">53.09 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">55.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">5.00 || <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">3.00 ||
 * **<span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">Product length ** |||||||||||||||||| <span style="color: #222222; font-family: Arial,sans-serif; font-size: 10pt;">138 ||

The above primers fell within the most conserved region of HIF-1a (NCBI BLAST):

=10/11/2011*=

Collected 16 English sole liver and heart tissue samples (32 total samples) from Hood Canal, WA. Hypoxia is currently very shallow, so there is a high chance of these English sole being exposed to low DO conditions.

All samples were placed in 5ml of RNAlater and stored at -20 o C. = = =7/28/2011***=

Made new batch of 1x TAE //20ml of 50x TAE// //980ml of NANO Pure H20//

Made new small (75ml of 1x TAE) 1.3% gel (1g of Agarose, 6ul of EtBR)

Ran 7/22/2011 PCR products and NTCs for (100V 30min)

= = Once again got bands in all four NTCs.....

Next step? Run only (four) NTCs using these primers and perhaps run a known, single banded primer & sample of a different species for comparison. Will use all new PCR H2O **__AND__** Apex Red.

=7/22/2011*=

Diluted new primers to 100uM



Ran conventional PCR for the two new sets of primers (protocol 7/8/2011)

//__MASTER MIX CALCULATIONS (__**__X2):__// //12.5ul 2xApex Red * 7 = 87.5ul// //0.5ul Forward primer * 7 = 3.5ul// //0.5ul Reverse primer * 7 = 3.5ul// //9.5ul PCR H20 * 7 = 66.5ul <- USED NEW BATCH OF H20 IN CASE PREVIOUS H20 WAS CONTAMINATED (i.e., NTC bands)//

//*Product stored at 4C for later gel run....// = = =7/15/2011***=

Ordered new primers ([|IDT]) of __known__ English sole sequences:

(GenBank EF119288) 16S ribosomal RNA gene, partial sequence; mitochondrial
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;"> **Primer pair 1** || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Sequence (5'->3') || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Strand on template || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Length || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Start || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Stop || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Tm || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">GC% ||
 * <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Forward || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">CCCGCCTGCCCAGTGACAAC || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Plus || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">20 || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">34 || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">53 || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">60.25 || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">70.00% ||
 * <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Reverse primer || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">CCATGGTCGCCCCAACCGAA || Minus || 20 || 327 || 308 || 59.34 || 65.00% ||
 * <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 110%;">Product length || 294 ||  ||   ||   ||   ||   ||   ||

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 110%;">(GenBank AF026744) GTPase K-rasB proto-oncogene mRNA, complete cds


 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 14px;">**Primer pair 10** || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Sequence (5'->3') || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Strand on template || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Length || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Start || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Stop || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Tm || <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">GC% ||
 * <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Forward || <span style="color: black; font-family: 'Times New Roman',serif; font-size: 13.5pt;">GGTGGGAGCTGGTGGCGTTG || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Plus || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 14px;">20 || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 14px;">20 || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 14px;">241 || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 14px;">60.18 || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 14px;">67.67% ||
 * <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Reverse primer || <span style="color: black; font-family: 'Times New Roman',serif; font-size: 13.5pt;">CCACTGTCCGGAACGGGAGGT || Minus || 21 || 593 || 573 || 60.18 || 66.67% ||
 * <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 14px;">Product length || 353 ||  ||   ||   ||   ||   ||   ||

[|link to Primer Search]

=7/13/2011***=

Ran gel for re-do Primer 9 samples (same protocol as below: 7/11/2011):



No good!

=7/11/2011***=

Ran two small agarose gels for both primer pairs.

//Used pre-existing gel for first run(Primer 3 samples)// //Mixed up a new batch for small gel mold (Primer 9 samples):// //75ml of 1x TAE// //1g of agrarose// //1ul of Ethedium Bromide (EtBr)//

Hyperladder 1 in the 1st well, 4 samples and 2 non-template controls (NTCs) in the remaining wells.

Ran both gels (separately) for ~30min at 100v

__Primer 3__: Unsuccessful :'o( __Primer 9__: Contaminated NTCs :o/ No pic.

Proceeded by redoing //Conventional PCR (2x Apex Red)// for Primer pair 9. NOTE: Same protocol and Master Mix proportions as below (7/8/2011) = = =7/8/2011*=


 * Conventional PCR was preformed on the sole cDNA.**


 * The following (Roberts Lab) protocol was followed:**

//Conventional PCR (2x Apex Red)//** Single reaction (25uL) set up is listed below. Be sure to make a master mix volume that will accommodate all of your samples, two water (no template controls; NTC) samples, plus an extra 10% to accommodate pipetting errors. Distribute appropriate amount of master mix (volume of master mix + template = 25uL) to PCR tubes or PCR plate. Make sure all tubes/caps are tightly closed. Put in thermalcycler. __//MASTER MIX CALCULATIONS (**X 2**)://__ //12.5ul 2xApex Red * 7 = 87.5ul// //0.5ul Forward primer * 7 = 3.5ul// //0.5ul Reverse primer// //* 7 = 3.5ul// //9.5ul PCR H20 * 7 = 66.5ul//
 * **Reaction_Components** || **Volume** || **Final Concentration** ||
 * 2x Apex Red || 12.5 || 1x ||
 * Forward Primer (10uM) || 0.5 || 0.2uM ||
 * Reverse Primer (10uM) || 0.5 || 0.2uM ||
 * Template || Up to 5uL ||  ||
 * H2O (PCR grade) || variable || Use to bring reaction volume up to 25uL ||



//NOTE: Did two separate batches of MM for each primer pair.//

Total of 12 PCR tubes: 2 H1 2 L1 2 H3 2 L2 4 NTC

Typical cycling paramaters (ask for help on using the thermal cycler):

95C - 10mins 40 cyles of: 95C - 15s 50-60C - 15s 72C - 10s - 2mins (dependent on amplicon size; ~1000kb/min)

Stored at 4C for running gel next week.... = = =7/6/2011***=

Reverse Transcribed sole RNA (i.e., cDNA)

The following (Roberts Lab) protocol was followed:

//Reverse Transcription (Promega M-MLV Protocol)//
A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.


 * 1) Use as much RNA as possible, max //volume// of RNA = 17.75uL. Generally, identify the RNA sample with the lowest concentration and multiply by 17.75uL. Use this //quantity// (ug) of RNA for each and every sample.
 * 2) Transfer calculated volume(s) of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O.
 * 3) Add appropriate amount of primer to sample. Use 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT in this example). Total volume (RNA + primers) should equal 18.25uL.
 * 4) Heat samples at 70C for 5 min in thermocycler.
 * 5) Place samples on ice IMMEDIATELY.
 * 6) Make __Master Mix__:

__PER RXN__ 5 uL 5x Buffer (M-MLV RT Buffer) 1.25 uL 10mM dNTPs 0.5 uL M-MLV RT **per ug of RNA**

7. Mix well. 8. Add 6.75uL of master mix to each reaction. 9. Mix well, but do not vortex. 10.Spot spin. 11.Incubate @ 42C for 1hr in thermalcycler for oligo dT primers OR @ 37C for random primers. 12.Heat inactivate @ 95C for 3 min. 13.Spot spin. 14.Store @ -20C.

__//MASTER MIX CALCULATIONS://__ //5ul M-MLV RT Buffer * 4.5 = 22.5ul// //1.25 dNTPs * 4.5 = 5.625ul --> rounded to 5.63ul// //0.5 M-MLV RT (per ug of RNA) * 4.5 = 2.25ul//



**6/30/2011*****



Successful RNA extraction of two heart and two liver samples of English Sole; apparent by the 260/280 ratios within 2 +/- 0.1.

The following (Roberts Lab) protocol was followed: NOTE: no DNase treatment

//RNA Extraction//
[|Manufacturers' Protocol] - MRC - Rinse in DEPC water in 50 ml falcon tube (3x – 3 separate tubes)
 * 1) Turn centrifuge on to cool to 4C
 * 2) Clean Homogenizer
 * 1) Get sample and thaw enough to get out of container
 * 2) Measure weight of sample
 * 3) Take sample out (screw out and use forceps) and chop up with sterile razor blade
 * 4) Put tri-reagent (stays on ice when not using & is light sensitive) into 50 ml falcon tube (or smaller tube depending on size of sample) – for a 0.7 gram sample I used 7 ml of tri-reagent. Note: in 50 ml falcon tube need at least 3 ml of tri-reagent to get it to work
 * 5) Add sample
 * 6) Keep on ice
 * 7) Blot homogenizer with paper towel to remove excess water
 * 8) Homogenize sample (don’t leave off ice for too long)
 * 9) Homogenize until sample is in solution
 * 10) Transfer all or part (I kept 6 ml) of mixture into a 13 ml tube (only add up to 7 ml)
 * 11) Let sit for 5 min at RT
 * 12) Rinse homogenizer in DEPC water (same tube used to clean in the beginning)
 * 13) Add 0.2 ml of chloroform (under hood, open only in hood, pour into glass beaker first) per 1 ml of tri-reagent
 * 14) Cover & shake
 * 15) Let sit for 15 min at room temp
 * 16) Change gloves
 * 17) Spin at 12,000 x g (11,500 rpm) for 15 min at 4C
 * 18) Transfer aqueous (top) phase to fresh tube (top layer has the RNA in it – bottom layer has DNA and proteins in it)
 * 19) Add 3 ml iso. (2 – Propanol, under hood) to precipitate out the RNA
 * 20) Cap and vortex
 * 21) Let sit at RT for 10 min
 * 22) Put waist with tri-reagent etc. in tri-reagent bottle in fume hood
 * 23) Clean up homogenizer and put away (put in 50 ml falcon tube with 5-7 ml 30% H2O2 and up to 40 ml with DEPC water
 * 24) Spin at 12,000 x g (11,500 rpm) for 15 min at 4C
 * 25) Remove supernatant (I want the pellet – RNA)
 * 26) Get glass beaker and paper towels (small stack)
 * 27) Pour off supernatant into beaker and place tube upside-down on paper towels
 * 28) Note: do not rock the tube back and fourth or will loosen pellet
 * 29) Add 1 ml 75% EtOH in DEPC water per 1 ml of tri-reagent added in beginning
 * 30) Cap & move – rock back and fourth to loosen pellet – vortex if necessary
 * 31) Spin 11,500 x rpm for 5 min at 4C
 * 32) Remove supernatant again & put on paper towel – be much more careful to make sure pellet does not slip out
 * 33) Spot Spin – turn on centrifuge, let go up to about 1000 rpm then shut off – note: place pellet facing upward
 * 34) Use filter pipette tips to remove excess EtOH
 * 35) Turn upside down on paper towels
 * 36) Wait 10 mins
 * 37) Depending on size of pellet add dnase free water to the tube – if taking to mRNA always use 500 ul (I used 500 ul for the ovary – large pellet, and 250 ul for the muscle)
 * 38) Dissolved into solution by pipetting
 * 39) Put in 1.5 ml tube
 * 40) Put on ice
 * 41) Spec to determine how much RNA you have

Also ordered ([|IDT]) 2 pairs of the following //Platichys flesus// (European flatfish) HIF-1a primers ([|GenBank: EF100709]) for PCR:

//**Primer Pair Three**// Sequence (5'->3') Strand on template Length Start Stop Tm GC% Forward primer GGCTCGGAGCGGAGGAAGGA Plus 20 37 56 60.04 70.00% Reverse primer TCCCGCAGCTCCTCCTGGTC Minus 20 440 421 59.97 70.00% Product length **404**

//**Primer Pair Nine**// Sequence (5'->3') Strand on template Length Start Stop Tm GC% Forward primer CCACAGCGTCAGCTCCAGCC Plus 20 138 157 60.04 70.00% Reverse primer TGACGTTGACAGTGCGGCCC Minus 20 553 534 59.91 65.00% Product length **416**