Jen's+Notebook

=__20130510__= .

Designed the following C.gigas primers for MeDIP PCR. All primers had at least 1 CG dinucleotide in them.
 * Primer name || Gene || Gene Accession# ||
 * Cg_MAPK_MeDIP_F || Mitogen-activated protein kinase 7 || CGI_10020574 ||
 * Cg_MAPK_MeDIP_R || Mitogen-activated protein kinase 7 || CGI_10020574 ||
 * Cg_CATHB_MeDIP_F || Cathepsin B || CGI_10027022 ||
 * Cg_CATHB_MeDIP_R || Cathepsin B || CGI_10027022 ||
 * Cg_CATA_MeDIP_F || Catalase || CGI_10003355 ||
 * Cg_CATA_MeDIP_R || Catalase || CGI_10003355 ||
 * Cg_CAP_MeDIP_F || Caspase - 3 || CGI_10015439 ||
 * Cg_CAP_MeDIP_R || Caspase - 3 || CGI_10015439 ||
 * Cg_SOD_MeDIP_F || Superoxide dismutase [Cu-Zn], chloroplastic || CGI_10025077 ||
 * Cg_SOD_MeDIP_R || Superoxide dismutase [Cu-Zn], chloroplastic || CGI_10025077 ||

= =

=__20130503__=

Ran qPCR on the following samples of C. gigas using primers Cg_HSP70_cm_f1 (SRID: 971) and Cg_HSP70_cm_r1 (SRID 972).

Single reaction (20uL) set up is listed below.

Mastermix was made of 10 uL 2X Sso Fast EvaGreen, 0.5 uL of 10 µM Forward Primer, 0.5 uL of 10 µM Reverse Primer, 8 uL Nano Pure Water.

Lane 1 = NTC Lane 2 = NTC Lane 3 = NTC Lane 4 = Exp 1 - 2, 1 uL Lane 5 = Exp 1 - 12, 1 uL Lane 6 = Exp 1 - 26, 1 uL Lane 7 = Exp 1 - 138, 1 uL Lane 8 = Exp 1 - 146, 1 uL Lane 9 = Exp 1 - 158, 1 uL

Centrifuged qPCR plate for 1 min @ 3000g.

Put in thermalcycler. C ycling paramaters:

1) 95C - 2 min 2) 98C - 2 sec 3) 55C - 5 sec 4) Plate Read 5) Go to line 2, 39 times 6) 75C - 10 sec 7) Melt Curve from 55C to 95C, read every 0.2C, hold 10 sec

Results: All samples amplified.

=__20130502__=

Discovered three different accession numbers for HSP70 in the Robert's Lab Database. Blasted all three separately. Each accession number matches almost perfectly with at least one other entry for HSP70 /HSP in GenBank. Sam said that the extremely high homology amongst HSP70 's in GenBank makes its use a bad idea. BLAST results are shown below:

https://www.evernote.com/shard/s256/sh/52fc77c3-74ae-426c-a966-1238329eca7c/0a9bfb1f4fcbbafd6c93b35593cc91ce

=__20130501__=

Prepared Reagents for MeDIP:

5x Buffer, 10 mL Solution 50 mM Na2HPO4 **(0.5 M Na2HPO4* X = 50 mM Na2HPO4 * 10 mL, X = 1 mL = __1000 uL, 0.5 M Na2HPO4)__** 700 mM NaCl **(5 M NaCl * X = 700 mM NaCl * 10 mL, X = 1.4 mL = __1400 uL, 5 M NaCl)__** 0.25% Triton-x 100 **(0.0025 * 10 mL = 0.025 mL = __25 uL, 0.25% Triton-x 100)__** [pipette slowly]

1x Buffer, 10 mL Solution
 * (5x Buffer * X = 1X Buffer * 10 mL, X = 2 mL) Use __2 mL of 5x Buffer + 8 mL H2O)__**

Digestion Buffer, 5 mL Solution 50 mM Tris HCl, pH = 8.0 **(100 mM Tris HCl * X = 50 mM Tris HCl * 5 mL, X = 2.5 mL = __2500 uL 100 mM Tris HCl)__** 10 mM EDTA, pH = 8.0 **(0.5 M EDTA * X = 10 mM EDTA * 5 mL, X = __100 uL, 0.5 M EDTA__** 0.5% SDS **(Prepared 50 mL of a 20% SDS Solution by combining 10 g SDS + 50 mL H2O; add 0.025 mL = __25 uL, 20% SDS)__** = = =__20130425__= **Results of PCR Performed on __20130424__**  Ran 20 uL of each C. gigas sample through agarose gel electrophoresis

TAE 100 mL Agarose 0.93 g EtBr 10 uL Hyper Ladder II, 5 uL Ran gel electrophoresis for 35 minutes at 120 volts.

Results:

All samples show high intensity bands ~95 bp.

https://www.evernote.com/shard/s256/sh/7c8097d1-be5b-42ba-bf33-03bf2c1331f8/15a3034dce69d4e1c7f5c2198fcda38a



Gel Layout:

Lane 1 - Hyper Ladder II Lane 2 - Negative Control Lane 3 - Negative Control Lane 4 - Negative Control Lane 5 - Exp 1 - 2 Lane 6 - Exp 1 - 12 Lane 7 - Exp 1 - 26 Lane 8 - Exp 1 - 138 Lane 9 - Exp 1 - 146 Lane 10 - Exp 1 - 158

The following results were given after running the primers Cg_HSP70_cm_f1 (SRID: 971) and Cg_HSP70_cm_r1 (SRID 972) through NCBI Blast:
 * Will post on google + to inquire about these blast results.

https://www.evernote.com/shard/s256/sh/ca72411b-db7c-46a9-841f-f22a5da7eec9/e80d21b310852255d57f2d2b359e6a5e

= = =__20130424__=

PCR - C. gigas
Performed Conventional PCR to amplify the following samples of C. gigas using primers Cg_HSP70_cm_f1 (SRID: 971) and Cg_HSP70_cm_r1 (SRID 972). Exp 1 - 2, 2 uL Exp 1 - 12, 2 uL Exp 1 - 26, 2 uL Exp 1 - 138, 2 uL Exp 1 - 146, 2 uL Exp 1 - 158, 2 uL Mastermix was made of 12.5 uL 2X Apex Red, 0.5 uL of 10 µM Forward Primer, 0.5 uL of 10 µM Reverse Primer, 9.5 uL Nano Pure Water with 2 µL DNA from C. gigas samples. Thermocycler profile was 95°C 10 min; 40 cycles of 95° for 30 sec, 55° for 30 sec, 72° 30 sec; 72° 10 min. Held overnight at 4°

Prepared Reagents for -MEDIP
MEDIP 5X Buffer was made of 0.8144 g Sodium Phosphate, 4.0766 g NACL, 25 mL Triton X, filled to 10 mL with water. MEDIP 1X Buffer was made of 4 mL water, 1 mL MEDIP 5X Buffer. Digestion Buffer was made of 5 mL water, 0.4006 g Tris HCL brought to 8.02 pH, 0.1436 g EDTA brought to 8.01 pH, and 0.0028 g SDS.

=__20130419__=

Examined the Quality of Extracted DNA from C. gigas, along with 6 other Sample From Emma
Ran the following samples through agarose gel electrophoresis with 4 ul of 5X Loading Dye : Exp 1 - 2, 5 uL Exp 1 - 12, 10 uL Exp 1 - 26, 15 uL Exp 1 - 138, 5 uL Exp 1 - 146, 5 uL Exp 1 - 158, 5 uL //C. gigas// - 101B2, 2 uL //C. gigas//- 101B5, 2 uL //C. gigas//- 101B8, 2 uL //C. gigas//- 103B224, 2 uL //C. gigas//- 103B227, 2 uL //C. gigas//- 103B230, 2 uL

TAE 200 mL Agarose 1.6 g EtBr 20 uL Hyper Ladder II 5 uL Ran gel electrophoresis for 35 minutes at 120 volts.

Results:

All samples displayed high molecular weight bands with no smearing.

https://www.evernote.com/shard/s256/sh/594e28e3-50ae-4fc7-9e1d-618ba1f8c089/8015eb899d1ec4171cce6928f60aaf2d



=__20130418__=

DNA Extraction - C. gigas (Gill?) Tissue Samples (Obtained From Emma)
Extracted DNA from the following Pacific Oyster samples using DNAzol: Exp 1 - 2 Exp 1 - 12 Exp 1 - 26 Exp 1 - 138 Exp 1 - 146 Exp 1 - 158


 * 1) Homogenized ~40 mg tissue in 1 mL DNAzol with a sterile pestle in a 1.5 mL sterile microfuge tube.
 * 2) Incubated samples at room temperature (RT) for 5 minutes.
 * 3) Centrifuged samples for 10 minutes at 10,000 g.
 * 4) Added 0.5 ml of 100% ethanol, then inverted tubes 8 times to form a homogeneous solution.
 * 5) Stored samples at RT for 60 minutes.
 * 6) Centrifuged samples for 5 minutes at 5,000 g.
 * 7) Removed supernatant with pipette, then washed DNA precipitate with 1.0 ml of 75% ethanol.
 * 8) Inverted tubes 5 times to form a homogeneous solution.
 * 9) Centrifuged samples for 2 minutes at 1,000 g.
 * 10) Repeated steps 7-9.
 * 11) Removed ~90% of the alcohol using a pipette, then centrifuged the samples for 2 minutes at 1,000 g.
 * 12) Removed remaining alcohol using a pipette and added 200 mL of Nano Pure H2O to each sample.
 * 13) Stored samples at RT for 25 minutes.

Quantified DNA using Nanodrop spectrophotometer, which gave the following results:

https://www.evernote.com/shard/s256/sh/caaaecba-c01f-4a13-b629-bd85d7916203/388d4173d163eac26060c132a347bb9c