Ran PCRs using both the AF133090 full-length primers (WS_16s_1_F, WS_16s_1501_R) and the WSN primers (WSN1_F, WSN1_R). This time used the linearized pCR2.1/AF133090 plasmid as template instead of the bacterial colonies that were selected from cloning. Master mix calcs are here. Cycling params are as follows:
95C – 10m
40 cycles of:
- 95C – 10s
- 55C – 10s
- 72C – 1.75m
Lane 1 – Hyperladder I (Bioline)
Lane 2 – 16s primers
Lane 3 – 16s primers NTC
Lane 4 – WSN primers
Lane 5 – WSN primers NTC
Essentially, this is the same result as the colony check from 20120503. The appropriate sized band (~1500bp) is generated using the 16s primers, but no band is generated using the WSN primers. Yes, there is contamination present in the 16s NTC sample, but this point is moot since the WSN primers still fail to generate a PCR product.
So, I have absolutely no idea what is cloned into this vector, despite the fact that primers designed on GenBank AF133090 produce a band.
I’m essentially running out of ideas on what else to do to get this working again. Will ask Lisa to make a curve from the linearized plasmid stock from DATE (not the pCR2.1/AF133090 plasmid that is being tested here) and see if she can get it to work. Also, Carolyn has suggested trying to run some of the more recent curves on a different qPCR machine. Will contact the guy in Health Sciences who has a Stratagene that we are allowed to use.