qPCR – Colleen’s Oyster cDNA 2GE with OsHV NGS Primers

A sample that was not in the strip of cDNA tubes was not qPCRd in the last run (20110518). Ran that sample (labelled 2GE R 1/5), as well as positive control DNA (07-CB-719-3), 1GE (cDNA) and 3GE (cDNA). Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF)

Overall, samples look good (no signal in NTCs, clean melt curves, reps are good). Data has been sent to Colleen for analysis.

qPCR – Test Colleen’s OsHV NGS Primers

Tested Colleen’s newly designed OsHV primers, which were based off of SOLiD next-gen sequencing expression data. Three primer sets (OsHV_ORF87, OsHV_ORF104, and OsHV_ORF107) were tested on two different DNA samples recommended by Colleen (07-CB-719-1 & 07-CB-719-3; both from 10/19/07). Master mix was set up according to Colleen’s protocol. Master mix can be found here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF).

Results show good amplification and clean melt curves with primers OsHV_ORF87 and 107. OsHV_ORF104 shows good amplification, but multiple peaks in the melt curve. Will run qPCR on cDNAs using primers OsHV_ORF87 and 107. Will discuss with Colleen and Carolyn whether they want to try optimizing the reaction for primer set OsHV_ORF104.