Tag Archives: PCR Miner

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.

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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.

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qPCR – COX qPCR Vibrio Exposure Response Check

Used COX primers (SR IDs 1060, 1061) and cDNA from 20080327, which consisted of 7 control gigas gill and 7 vibrio-exposed (24hrs) gigas gill samples, labeled as C# and VE#, respectively. The experiment was a 24hr. exposure live Vibrio vulnificus, parahaemolyticus Cf = 2.055×10^11 (6.85×10^7 Vibrio cells/oyster).
Note: Used a free sample of 2x Brilliant III Ultra Fast SYBR Green QPCR Master Mix (Stratagene) for this qPCR. Mixed components and set up cycling params according to the manufacturer’s recommendation for the BioRad CFX96.

Master mix calcs are here. Plate layout, cycling params, etc. can be see in the qPCR Report (see Results).

Results:

qPCR Report (PDF).

PCR Miner analysis is here. There appears to be an increase in COX expression in samples exposed to Vibrio sp. (see graph below), however, I have not determined if the results are significant.

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