This is an exact repeat of the PCR from yesterday, but with a brand new vial of Apex Red Master Mix, in an attempt to eliminate the contamination previously seen in the NTCs.
Ladder: Hyperladder I (Bioline)
Well, for some reason there are still bands in the NTCs. However, they appear to be of different sizes than the bands in the clam DNA samples. I think they’re OK to use and the cloning/sequencing is cheap enough these days, that I’ll just get these sequenced and see what we have.
I excised each of the bands in the clam DNA samples (16s = ~2000bp; EUB = ~2100bp) and purified them using Ultrafree-DA spin columns (Millipore) in preparation for cloning.
After the last PCR continued to exhibit products in the no template controls (NTC) for most of the primer sets I was using, I ordered new primers. They arrived today so, I re-ran the PCR on the clam RLO DNA isolated 20150130 with the following new primers:
Master mix calcs are here: 20150223 – cPCR Universal Primers Apex Red MM
Cycling params were:
1 cycle of:
40 cycles of:
- 95C – 15s
- 50C – 15s
- 72C – 1min
Samples were run on 1.0% agarose, low TAE gel stained w/EtBr.
Ladder: Hyperladder I (Bioline).
Crazy; contamination still present in the NTCs. Primer stocks were steriley reconstituted with Low TE Buffer (IDT) and working stocks were created steriley, so I’m not really sure why this is continuing to happen. Possibly the polymerase is contaminated? Will try again with previously unopened polymerase and see how that plays out.
No bands were excised since I can’t be certain that the bands present in the Clam DNA samples are from the actual sample and not from the apparent contamination.