Rony's+Notebook

6/04/2010 Master Mix for PCR: cDNA 1 ul go Taq 12.5 ul Pf .5ul Pr .5ul H20 10.5

95 degree - 10min 95 degree - 15sec - 40x 55 degree - 15sec - 40x 72 degree - 1min - 40x

6/01/2010 Prep PCR Working Stock: 10uL of Primer + 90uL Water

5/28/2010 Reverse Transcription on Acid shock fish sample. Sample: 6.2.1-6.2.8, 6.7.1, 6.7.3-7.7.8, 6.10.1-6.10.4, 6.10.6, 6.10.8 Spreadsheet for the sample [|Acid Shock Spreadsheet] Master Mix: 5 uL 5x Buffer (M-MLV RT Buffer) x24 = 120 ul 1.25 uL 10mM dNTPs x24 = 30 0.5 uL M-MLV RT per ug of RNA x24 = 12

5/11/2010 RNA isolation from pH treated fish

5/4/2010 RNA isolation from pH treated fish

4/15/2010 Isolated Plasmid from Oyster bacteria.

4/6/2010 Can't merge files using galaxy because galaxy is not reading the ''Herring selected file" as data so this is not listed as an option when trying to join the files together.

3/8/2010 Extract RNA and Spec'd samples

3/1/2010 Extract RNA and Spec'd the samples

2/25/2010 Extract RNA (per tube):
 * 1) 500ul of Tri-reagent in RNA tube
 * 2) Add severed fish head and grind as much as possible
 * 3) Transfer 100ul into another RNA tube
 * 4) Then add 900ul of Tri-reagent to get to 1ml
 * 5) Follow the RNA Protocol
 * 6) Take the aqueous out and spin for a couple of seconds to separate.
 * 7) Get as much aqueous out
 * 8) Then add 30ul of +H2O
 * 9) Spec'd the samples



1/25/10 4hrs 1/

1/14/10 3.5hrs data entering for Mac

1/13/10 2hrs

1/11/2010 4.5hrs Worked on Paper Set alarm on fridge

1/6/2010 2 hrs Worked on Paper

1/5/2010 2hrs Worked on Paper

1/4/2010 Dish washing Checking Pipette accuracy

12/8/09 4hrs Work on GIS

12/7/09 3hrs Work Paper

12/3/09 5hrs Purified DNA for sequencing.
 * 14.8 C || Bacteria || Oyster ||
 * Control || 7.63 || 7.08 ||
 * CO2 || 7.19 || 7.29 ||

11/30/09 Run PCR for the remaining samples
 * 15.2 || Bacteria || Oyster ||
 * Control || 7.69 || 7.14 ||
 * CO2 || 6.93 || 7.24 ||

11/24 3hrs Made gel for PCR 150uL of tae buffer 1.5g agar 1.5uL ethidium bromide Added extra water

Top (left to right): A8, H7, G7, F7, E7, D7, C7, B7, A7, H6, G6, F6, E6, D6, C6, B6, A6, H5, G5, Ladder Bottom (left to right): D10, C10, B10, A10, H9, G9, F9, E9, D9, C9, B9, A9, H8, G8, F8, E8, D8, C8, B8, Ladder Measured pH for Oyster and Bacteria
 * 14.5 C || Bacteria || Oyster ||
 * Control || 7.61 || 7.18 ||
 * CO2 || 7.06 || 7.21 ||

11/23 4 hrs Made gel for PCR 150uL of tae buffer 1.5g agar 1.5uL ethdium bromide

Run PCR Top( from left to right) Ladder, A1, B1, C1, D1, E1, F1, G1, H1, A2, B2, C2, D2, E2, F2, G2, H2, A3, B3, C3 Ladder, D3, E3, F3, G3, H3, A4, B4, C4, D4, E4, F4, G4, A5, B5, H4, C5, D5, E5, F5

Measured pH level of Oyster and Bacteria.
 * 16.2 C || Bacteria || Oyster ||
 * Control || 7.65 || 7.16 ||
 * CO2 || 6.86 || 7.23 ||

11/19 5.5hrs Measured pH level for CO2 affects on Oyster and Bacteria. Spin samples for bacteria and freeze them at 20 C. Prepare samples of Lake Trout Population for PCR. Master mix DNA - 1uL 2x Apex - (12.5 x 14) = 175uL Pf/Pr - 7uL H2O - (10.5 x 14) = 147 uL
 * || Bacteria || Oyster ||
 * Control || 7.56 || 7.28 ||
 * CO2 || 6.96 || 7.18 ||

Incubator 95 degree 10mins 95 degree 10mins (X40) 60 degree 15 sec (X40) 72 degree 25 sec (X40) 72 degree 10 mins

11/17 2.5hrs Set up 24 well electroylsis 150 mL of TAE buffer 3 g agar (need to within 2% of TAE buffer) 15 uL of ethdium bromide (1 uL for every 10mL)

Set up electrolysis gel (large), USE GLOVES! 1. 150 ml of TAE buffer 2. 3 g agar 3. heat for 3 min 4. add 11.48 g of water 5. wait 15 mins 6. add 15 ul of ethdium bromide (fridge) 5. pour into plate, rest for 35 min
 * ~ Top (left to right) || x || Con(6) || CO2(6) || x || Con(7) || CO2(7) || x || Con(9) || CO2(9) || x || Con(13) || CO2(13) || x || Con(14) || CO2(14) || x || Con(15) || CO2(15) || x || Ladder ||
 * ~ Bottom (left to right) || x || Con(19) || CO2(19) || x || Con(38) || CO2(38) || x || Con(20) || CO2(20) || x || Con(18) || CO2(18) || x || Con(17) || CO2(17) || x || Con(12) || CO2(12) || x || Ladder ||
 * Note - Added 20ul of Ladder for each slot, 7uL for each sample, and 30ul at of ethdium bromide

Oyster Tank pHs user:kubu4 Right Front - 7.39 Left Front - 6.85 Right Back - 7.26 Left Back - 7.05
 * || Bacteria || Oyster ||
 * Control || 7.39 || 7.26 ||
 * CO2 || 6.85 || 7.05 ||

11/16 3hrs Cleaned container for the CO2 project Worked on Papers

11/12 5hrs Link Colton pics Work on Mac's data Download articles into Papers

11/10 3.5hrs Work on GIS Input data for Mac

11/9 3hrs Link Colton pics to this page Work on GIS

11/2 3hrs Download anything related to Puget Sound

10/29 6hrs Install ArcGIS and play around with it

10/27 2hrs Import articles to Papers

10/26 3.5hrs Import articles to Papers

10/20/2009 2hrs Import articles to Papers

8/14/2009 5hrs Link colton pictures to website

8/13/2009 3hrs Colton pictures

8/12/2009 3hrs Colton pictures

8/10/2009 4hrs Backup wiki

The result of PCR from last week sample. . From top left is Water, Water, Sea Male #1, Bay, 7, 6, 5, 4, 3, 2, 1 and ladder. At the bottom is Water, Water, Sea Male #2, Sea,, 14, 13, 12, 11, 10, 9, 8 and ladder. Since this PCR used the primer Sea RV 2, the two sea male samples are positive. From the previous PCR with Bay reverse primer, all but one sample was positive. Meaning that all but one were Bay scallops and the negative sample, #13, was a Sea scallops. So the expected result for this gel should have only sample #13 that is positive while everything else should be negative. The actual result have everything as negative even sample #13. So something went wrong with sample #13, it should've been a positive for the PCR with the Bay reverse primer or positive with the Sea primer. Beside from that the rest of the result was expected.

8/5/2009 3hrs Incubator 95 degree 10mins 95 degree 10mins (X40) 56 degree 1min (X40) 72 degree 90 sec (X40)

Redo PCR for Hybrid samples with Sea Actin RV 2. Also Sea scallop male 1 and 2 were used for this PCR run.

The gel result of yesterday PCR. Something went wrong because every slot is negative.

8/4/2009 3hrs Prepare PCR for Hybrid samples with Sea Actin RV 2 Each sample consist (except water samples) of 2uL of gDNA, 12.5uL Go Taq, .5uL Scallop Actin FW, .5uL Sea Actin and 9.5uL water. Water samples have an extra 2uL of water to replace the gDNA amount.

Incubator 95 degree 10mins 95 degree 10mins (X40) 56 degree .30 sec (X40) 72 degree 1min (X40)

8/3/2009 Run PCR for Hybrid samples Top row (left to right) Water Water Sea 7 6 5 4 3 2 1 Ladder Bottom row Water Water Bay 14 13 12 11 10 9 8 Ladder From this gel, it seemed that everything is a Bay scallop except for one, sample #13. Since I used the Bay primer, everything that reacted with the primer, the positive, was Bay scallop.

Incubator 95 degree 10 mins 95 degree 10 mins (X40) 53 degree .30 sec (X40) 72 degree 1min (X40)

Prepare PCR for Hybrid samples with Bay Actin RVO Each sample consist (except water samples) of 2uL of gDNA, 12.5uL Go Taq, .5uL Scallop Actin FW, .5uL Bay Actin RVO and 9.5uL water. Water samples have an extra 2uL of water to replace the gDNA amount.
 * Sample || gDNA || 2x Go Taq || Scallop Actin FW || Bay Actin RVO || Water ||
 * 1 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 2 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 3 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 4 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 5 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 6 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 7 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 8 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 9 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 10 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 11 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 12 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 13 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * 14 || 2 || 12.5 || .5 || .5 || 9.5 ||
 * Sea Scallop || 2 || 12.5 || .5 || .5 || 9.5 ||
 * Bay Scallop || 2 || 12.5 || .5 || .5 || 9.5 ||
 * Water || 0 || 12.5 || .5 || .5 || 11.5 ||
 * Water || 0 || 12.5 || .5 || .5 || 11.5 ||
 * Water || 0 || 12.5 || .5 || .5 || 11.5 ||
 * Water || 0 || 12.5 || .5 || .5 || 11.5 ||

Note: To find 2x Go Taq eq: C1*V1=C2*V2

Set up 12 well electroylsis 150 mL of TAE buffer 1.5 g agar (need to within 1% of TAE buffer) 15 uL of ethdium bromide (1 uL for every 10mL)

Set up electrolysis gel (small), USE GLOVES! 1. 100 ml of TAE buffer 2. 1 g agar 3. heat for 3 min, mixing every min 4. add 6 ul of ethdium bromide (fridge) 5. pour into plate, rest for 35 min

7/31/2009 Updated species doc on google Prepared sample of hybrid scallops and used chelax on sample

7/30/2009

7/17/2009 4hrs Chelex seaweed and cocopods

7/13/2009 2.5hrs Update primerdatabase Link pictures to colton collection

6/17/2009 3.5hrs Took colton pictures

6/16/2009 3hrs Link colton pictures to genefish Fixed the colton collection page

6/12/2009 Journal of Shellfish Research June 2002-April 2009 Marine Biotechnology Feb 2004-June 2009 Journal of the World Aquaculture Society March 2002-March 2005 World Aquaculture March 2008-March 2009

6/5/2009 Took colton pictures

5/22/2009 2hrs Took Colton Pictures.

5/21/2009 5.5hrs Continue with citation and worked on ocean acidification

5/19/2009 5.5hrs Took pictures for colton collection Did citation for article

5/14/2009 4.5hrs Did Realtime PCR for Oysters Dermo sample Worked on ocean acidification sheet

5/12/2009 5hrs Made new sample for the Dermo experiment. Filled in ocean acidification sheet

5/7/2009 3hrs Set up Realtime PCR for Oysters Dermo experiment. Filled in ocean acidification sheet

5/5/2009 4hrs Spect the oyster samples Input data of oyster collection on google

5/1/2009 4hrs Continued ethanol precipitation for oyster sample.

4/30/2009 4.5hrs Spect the oyster samples and did ethanol precipitation for the samples. Mixture for each sample: DNA 100 mL 3M NaOAc 10 mL 100 l EtOH 220 mL Incubate 20c

4/28/2009 3.5hrs Took more photos for the colton collection and upload them. Filled in ocean acidification sheet on google.

4/23/2009 3hrs Looked for information about ocean acidification on shellfish or invertebrates.

4/22/2009 4hrs Set up Realtime PCR for Oysters Dermo experiment. [|Here's the set up of the samples]

4/21/2009 2.5hrs Uploaded recent photos to colton page and looked at oyster samples.

4/14/2009 3hrs Took more pictures for colton pictures Started the education database on google doc

4/10/2009 user:kubu4 Ran Ronnie's PCR samples on a gel. Lane 1 - 100bp ladder Lane 2 - Sea Lane 3 - Sea Rv H2O Lane 4 - Bay Lane 5 - Bay Rv.2 H2O Lane 6 - B x S, Bay Rv Lane 7 - B x S, Sea Rv Lane 8 - S x B, Bay Rv. Lane 9 - S x B, Sea Rv.

4/8/2009 Run PCR for Bay/Sea Scallop with Scallop Actin Pf Lane 1 Ladder Lane 2 Bay, Bay_Rv2 Lane 3 H2O, Bay_Rv2 Lane 4 Sea, Sea_Rv3 Lane 5 H2O, Sea_Rv3 There are no bands in the water sample so that means nothing is contaminated. Sea and Bay gDNA reacted with their reverse primer so they have bands in their lanes. But Sea and Bay band appear in different spot of the gel, this could be used to identify the two species.

Set up PCR for Bay/Sea Scallop Hybrid with Scallop Actin Pf 95 C - 10 min. 95 C - 45 sec. 55 C - 45 sec. 72 C - 1.30 min. 72 C - 10 min. 4 C
 * Test Tube || gDNA || 2x GoTaq || Pf || Pr || H2O ||
 * Bay, Bay_Rv2 || 4ul || 12.5 || 1 || 1 || 6.5 ||
 * Bay x Sea, Bay_Rv2 || 4ul || 12.5 || 1 || 1 || 6.5 ||
 * Bay x Sea, Sea_Rv3 || 4ul || 12.5 || 1 || 1 || 6.5 ||
 * Sea, Sea_Rv3 || 4ul || 12.5 || 1 || 1 || 6.5 ||
 * Sea x Bay, Bay_Rv2 || 4ul || 12.5 || 1 || 1 || 6.5 ||
 * Sea x Bay, Sea_Rv3 || 4ul || 12.5 || 1 || 1 || 6.5 ||
 * H2O, Bay_Rv2 || 0 || 12.5 || 1 || 1 || 10.5 ||
 * H2O, Sea_Rv3 || 0 || 12.5 || 1 || 1 || 10.5 ||

4/7/2009 Set up PCR for Bay/Sea Scallop with Scallop Actin Pf
 * Test Tube || 1 - Bay, Bay_Rv2 || 2 - H2O, Bay_Rv2 || 3 - Sea, Sea_Rv3 || 4 - H2O, Sea_Rv3 ||
 * gDNA || 4 ul || 0 ul || 4 ul || 0 ul ||
 * 2x GoTaq || 12.5 ul || 12.5 ul || 12.5 ul || 12.5 ul ||
 * Pf || 1 ul || 1 ul || 1 ul || 1 ul ||
 * Pr || 1 ul || 1 ul || 1 ul || 1 ul ||
 * H2O || 6.5 ul || 10.5 ul || 6.5 ul || 10.5 ul ||

4/1/2009 Work on OpenOffice database

Run PCR for Bay/Sea Scallop Lane 1 Ladder Lane 2 Bay Rv.2 Lane 3 Bay H20 Lane 4 Sea Rv.3 Lane 5 Sea H20

3/13/2009 Play with OpenOffice and entered some database

Set up PCR for Bay/Sea Scallop Test Tube


 * || __**1 - Bay, Bay_Rv2**__ || __**2 - H2O, Bay_Rv2**__ || __**3 - Sea, Sea_Rv3**__ || __**4 - H2O, Sea_Rv3**__ ||
 * gDNA || 1 || 0 || 1 || 0 ||
 * 2x GoTaq || 12.5 || 12.5 ul || 12.5 ul || 12.5 ul ||
 * Pf || 1 || 1 ul || 1 ul || 1 ul ||
 * Pr || 4 || 0 ul || 4 ul || 0 ul ||
 * H2O || 6.5 || 10.5 ul || 6.5 ul || 10.5 ul ||

95 C - 10min. 95 C - 45sec. 50 C - 45sec. 72 C - 1.5min. 72 C - 10min.

3/12/2009 Run PCR for Kelvin's Bay/Sea Scallop

Lane 1 Ladder Lane 2 Bay Lane 3 Sea Lane 4 Bay (Female) x Sea (Male) Sea Rv Lane 5 Bay (M) x Sea (F) Bay Rv Lane 6 Bay (M) x Sea (F) Sea Rv Lane 7 Bay (F) x Sea (M) Bay Rv Lane 8 H2O Sea Rv Lane 9 H2O Bay Rv

3/11/2009 Scan Papers Took colton pictures

3/6/2009 Run PCR - Remainder Bay/Sea Scallop samples Lane 1 Ladder Lane 2 Bay Actin Primer Reverse with H2O Lane 3 Bay Actin Primer Reverse with Bay x Sea Scallop gDNA Lane 4 Bay Actin Primer Reverse with Bay Scallop gDNA

Results: Bay Reverse Primer reacted with Bay gDNA but not with hybrid. There's nothing going on in Lane 2 with the water sample.

3/5/2009 Took shellfish pictures Run PCR - Bay/Sea Scallop samples (From the left) Lane 1 Ladder Lane 2 Sea Actin Primer Reverse with Sea Scallop gDNA Lane 3 Sea Actin Primer Reverse with Bay Scallop gDNA Lane 4 Sea Actin Primer Reverse with H2O Lane 5 Sea Actin Primer Reverse with Sea x Bay Scallop gDNA Lane 6 Sea Actin Primer Reverse with H2O Lane 7 Sea Actin Primer Reverse with Bay x Sea Scallop gDNA Lane 8 Sea Actin Primer Reverse with H2O Lane 9 Bay Actin Primer Reverse with Sea Scallop gDNA Lane 10 Bay Actin Primer Reverse with H2O Lane 11 Bay Actin Primer Reverse with H2O Lane 12 Bay Actin Primer Reverse with Sea x Bay Scallop gDNA

Results: The Sea Reverse Primer didn't react with the Sea Scallop DNA but it reacted with Bay Scallop DNA which is very unusual. Sea Reverse Primer didn't react with the hybrid samples. There is also something in the water for Lane 10 and 11 but for the other two lanes of water it looks normal.

2/27/2009

PCR - Bay/Sea Scallop samples

[|PCR work up here].

95 C - 10 min. 95 C - 45 sec. 55 C - 45 sec. 72 C - 1 min. 72 C - 10 min. 4 C

2/20/2009 Took shellfish collection pictures Label photos

2/19/2009 Took snapshot of shellfish collection Worked on shellfish collection Reconciled paper Did snp

2/18/2009 Took snapshot of shellfish collection

2/13/2009 Worked on fish collection

2/12/2009 Worked on snp form, end at 1234 so anything above 1234 is not done yet Worked on fish collection

2/11/2009 Worked on snp form Reconciled paper Worked on fish collection

2/6/2009 Worked on fish collection

Worked on the fish collection**
 * 2/5/2009

Learned how to reconciled paper and work the autoclave. Did some reconciling and scanning.
 * 2/4/2009**