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 – Colleen’s Oyster cDNA with OsHV NGS Primers

Ran qPCR with OsHV_ORF87 and 107 primer sets, based off successful test qPCR on DNA from earlier today (see below). qPCRs were run on cDNAs from Colleen’s #26 Box (in -20C in FSH 236) labeled 1GE – 9GE, 1GC, 3GC – 9GC. 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, most reps are good). However, I don’t know what these samples are, so I can’t comment on what the actual data implies in regards to this experiment. Data has been sent to Colleen.

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.

qPCR – Repeat of phage qPCR

This is a repeat from 20110329, due to poor results and contamination in the NTC samples. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF)

This turned out better than the qPCR done on 20110329. However, the AbV03 primer set results are still not totally consistent across reps. But, we do see amplification only in the 10:25-24 sample and not the other two. As of right now, the phage load in these samples has not been quantified with histology, so I can’t say how these results compare as to what we might expect to see (based on phage load). Additionally, the AbV06 primer set still failed to produce a product. Will discuss with Carolyn to see what she thinks…