PCR – Hexokinase Promoter and CDS (repeat from 20130227)

Performed a repeat of the failed PCR from 20130227, but used a pool of cDNA (made from 20110311 C.gigas cDNA) instead of a single sample and changed the annealing temp to 50C.

Results:

Same exact results as 20130227; nothing. As such, didn’t take gel image. Will retry one more time using a long-distance polymerase, along with varying [MgCl2].

UPDATE 20130318: Doh! When talking about this at lab meeting today, I realized I’m trying to amplify the promoter (a genomic sequence) using cDNA! Will re-design primers and develop new cloning strategy for this!

PCR – Hexokinase Promoter and CDS

Performed PCR to amplify the C.gigas hexokinase (ACCESSION#) promoter region (-2059bp) and the CDS without the stop codon. Elimination of the stop codon allows for subsequent cloning into the pBAD-TOPO expression vector, which will incorporate the V5 epitope tag sequence. This tag will be used to distinguish between endogenous hexokinase expression and expression generated from our hexokinase construct.

Used hexokinase primers (SR IDs: 1518, 1519).

Master mix calcs and cycling params are here.

Results:

Ladder: Hyperladder II (Bioline)

No amplification in sample or no-template control. Will re-do with lower annealing temp (50C).

Reverse Transcription – Herring RNA from 20091026

Performed an RT reaction on pooled herring gonad and liver mRNA from 20091026 for James Raymond at the UNLV. A single RT reaction was performed using 12.75uL (208ng) of the pooled gonad mRNA and 5uL (132.5ng) of the pooled liver RNA, according to our default MMLV (Promega) protocol. After reaction was completed, sample was stored @ -20C and then shipped to James Raymond on 20130214.

qPCR – Halley cDNA Check

Ran qPCR on Halley’s cDNA to see if I could get them to work. She has been getting high levels of fluorescence at the initiation of the qPCR cycling that shouldn’t be there. Master mix calcs and plate layout can be seen here. http://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20121128%20qPCR%20Layout.jpg

Cycling params can be found in the qPCR Data File (see Results).

Results:

qPCR Data File (Opticon2)

http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121128_111905.tad

High levels of initial fluorescence are present in both sets of cDNA samples, while the NTC sample does not exhibit this behavior, suggesting the template is to blame. Have suggested to Halley to make new cDNA using the correct recipe, instead of the FISH441 recipe she had been using.

Reverse Transcription – FISH441 RNA

Reverse transcribed the class FISH441 RNA to cDNA. Followed protocol provided on the FISH441 Wiki page, NOT the usual Roberts Lab protocol, with some modifications.

Changed total reaction volume to 50uL to accommodate volume of RNA, as well as low concentration of dNTPs that were to be used.

RNA – 17.75uL

Oligo dT – 1.0uL

Samples were heated at 70C for 5mins and placed immediately on ice.

Reaction buffer master mix calcs:

5x MMLV Buffer – 10uL/rxn X 88 = 880uL

2.5mM dNTPs – 10uL/rxn X 88 = 880uL

M-MLV – 1uL/rxn X 88 = 88uL

H2O – 9.25uL x 88 = 902uL

Added 31.25uL of the reaction buffer master mix to each well of RNA/Oligo dT. Reaction was run at 42C for 1hr, followed by 70C for 3mins. Samples were stored @ -20C in Rm. 209.

qPCR – Manila Clam Larvae cDNA (from August 2012 – Dave’s Notebook)

Ran qPCR on manila clam larvae cDNA that Dave made on 8/7/2012, using the sample sets from 7/29/2011 and 8/5/2011 of the OA manila clam experiment he ran.

Primers used:

Rp_Cathepsin_F/R2 (SR IDs: 1461, 1473)

Rp_EF1a_F/R2 (SR IDs: 1463, 1474)

Primers were verified to be in good working order by Dave on 4/1/2012 (see Dave’s notebook).

Master mix calcs are here. Cycling params can be found in the qPCR Data File (see Results). Plate layout and PCR Miner analysis can be found in the qPCR Raw Data file (see Results). All samples run in duplicate.

Results:

qPCR Data File(Opticon 2) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_172259.tad

qPCR Raw Dat and PCR Miner Analysis(Excel) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_172259.xlsx

Reps look pretty good, although the 4C2 8.5.11 sample has consistently bad reps across all of today’s runs. Data will be shared with Steven for comparison to Dave’s Illumina data.

All data was normalized to EF1a expression from this run.

qPCR – Manila Clam Larvae cDNA (from August 2012 – Dave’s Notebook)

Ran qPCR on manila clam larvae cDNA that Dave made on 8/7/2012, using the sample sets from 7/29/2011 and 8/5/2011 of the OA manila clam experiment he ran.

Primers used:

Rp_Calmodulin_F/R2 (SR IDs: 1449, 1467)

Rp_Crumbs_F/R (SR IDs: 1477, 1476)

Primers were verified to be in good working order by Dave on 4/1/2012 (see Dave’s notebook).

Master mix calcs are here. Cycling params can be found in the qPCR Data File (see Results). Plate layout and PCR Miner analysis can be found in the qPCR Raw Data file (see Results). All samples run in duplicate.

Results:

qPCR Data File(Opticon 2) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_161738.tad

qPCR Raw Data and PCR Miner Analysis (Excel) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_161738.xlsx

Reps look pretty good, although the 4C2 8.5.11 sample has consistently bad reps across all of today’s runs. Data will be shared with Steven for comparison to Dave’s Illumina data. All data was normalized to EF1a expression from later today.

qPCR – Manila Clam Larvae cDNA (from August 2012 – Dave’s Notebook)

Ran qPCR on manila clam larvae cDNA that Dave made on 8/7/2012, using the sample sets from 7/29/2011 and 8/5/2011 of the OA manila clam experiment he ran.

Primers used:

Rp_GPX3_F/R2 (SR IDs: 1453, 1469)

Rp_HSP90_F2/R2 (SR Ids: 1457, 1471)

Primers were verified to be in good working order by Dave on 4/1/2012 (see Dave’s notebook).

Master mix calcs are here. Cycling params can be found in the qPCR Data File (see Results). Plate layout and PCR Miner analysis can be found in the qPCR Raw Data file (see Results). All samples run in duplicate.

Results:

qPCR Data File(Opticon 2) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_150936.tad

qPCR Raw Data and PCR Miner Analysis(Excel) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_150936.xlsx

Reps look pretty good, although the 4C2 8.5.11 sample has consistently bad reps across all of today’s runs. Data will be shared with Steven for comparison to Dave’s Illumina data.

All data was normalized to EF1a expression from later today.

qPCR – DNased Manila Clam Larvae RNA (from August 2012 – Dave’s Notebook)

Performed qPCR on Dave’s manila clam larvae DNased RNA from August 2012 using EF1a primers (SR IDs: 1463, 1474).

Master mix calcs are here. https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdHc5amwzZzdDa1d0VXQzLVU0WkFTc0E

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Positive control was pooled cDNA taken from Dave’s cDNA plate on 8/7/2012.

Results:

qPCR Data File(CFX96) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pcrd

qPCR Report(PDF) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pdf

Here’s a quick Google Spreadsheet summary highlighting samples that came up positive/negative.

https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdFFHb3YwWE01UG00TnY3OWo2cWx2UVE

Approximately half of the samples (~27) came up positive for still having gDNA in them.

There are three pCO2 treatments: 1000ppm, 750ppm, and 400ppm. There are six sampling dates: 7/29/2011, 8/2/2011, 8/9/2011, 8/12/2011. Currently, it is unknown when the Day 0 samples were collected. Have emailed Dave for deets.

There are only two dates (7/29/2011 and 8/5/2011) that have a full set of samples (i.e. 1000ppm, 750ppm and 400ppm) that exhibit DNA-free RNA. Will discuss with Steven on how to proceed.

UPDATE 20121031 – Dave emailed and indicated the experimented started on 7/27/2011. Additionally, the two sample sets that are complete are Day 2 and Day 7. Discussing with Steven, we have decided to run a few genes and see how the expression levels compare to the NGS data analysis for these samples. If the qPCR data supports the NGS data, then that information will be relayed to the BMC Genomics reviewers in response to their critiques. A copy of the manuscript is here(may not be publicly viewable). https://docs.google.com/document/d/1Ii1lODz2oThiyxZtHBblUEdzyhIVq92n8jkEjhkuuts/edit

Received oysters from Taylor Shellfish.

Photos of oysters are here:

Bag labels and oyster quantities are below:

Hood Canal Dabob (Long Spit) 2012 Broodstock O. lurida PSRF – 42 oysters

Dabob Progeny (Long Spit) outplanted in Port Gamble Bay 8-20-2012 – ~97 oysters

O. lurida 2012 Seed Fidalgo Bay PSRF – Too small to count; settled on old oyster shells

O. lurida 2012 Broodstock Fidalgo Bay PSRF – 104 oysters

All oysters were put in our recirculating system in MAR 189 and held at 11C.