After previously confirming that the issue with previous PCRs was due to bad reagents, I re-ran the PCR on the clam RLO DNA isolated 20150130 using a set of universal 16s primers, as well as a universal 18s primer set to serve as a positive control that amplifiable DNA was present in the sample.
Master mix calcs are here: 20150219 – cPCR Universal Primers Apex Red MM
Primers being used are:
- 27F, 1492R
- EHR16D, EHR16R (universal ehrlichia)
- 18s EUK 581 F, 18s EUK 1134 R
Cycling params were:
1 cycle of:
- 95C – 10mins
40 cycles of:
- 95C – 15s
- 50C – 15s
- 72C – 1mins
Samples were run on 1.0% agarose, low TAE gel stained w/EtBr.
Ladder used was O’GenRuler 100bp DNA Ladder (Thermo-Fisher).
No sample was loaded directly next to ladder to facilitate excision, if necessary.
Each sample was accompanied by a no template control (NTC).
The ehrlichia universal primers (EHR) and the universal 18s (18s) primers are the only two primer sets that do not have contamination present in the NTCs.
Excised the EHR band and purified with Ultrafree-DA columns (Millipore). Purified DNA was stored @ -20C and will be used for cloning/sequencing next week.
Have already ordered additional primer sets of those above that are contaminated. Will re-run the PCR with those new, sterile primer sets when they arrive to obtain a larger product (the EHR amplicon is only ~350bp).