Since the previous check of the various universal primers with abalone DNA (sample 09:8-20) failed to amplify, even with withering syndrome primers, I’m testing repeating that PCR using a newer/different PCR master mix.
Template DNA is: 09:20-08 (from tissue)
Background info for template DNA is here: Red/Pink/Pinto
Primers being used are:
- 27F, 1492R
- EHR16D, EHR16R (universal ehrlichia)
- 18s EUK 581 F, 18s EUK 1134 R
- WSN1 (withering syndrome)
Master mix calcs are here: 20150212 – cPCR Universal Primers 09:8-20 Apex Red MM
All samples were run in duplicate.
Cycling params were:
1 cycle of:
- 95C – 10mins
40 cycles of:
- 95C – 15s
- 50C – 15s
- 72C – 2mins
Ran samples out on a 0.8% agarose, 1x TBE gel w/EtBr
Well, this is a good result. It demonstrates that the previous reagents that I had been using are no good. The primers work. However, it does appear that all of the universal primers (excluding the 18s and EHR) are contaminated. All of these primer sets were stocks that were prepared by other people and none of them were marked as being sterile (which they should be). Regardless, I’ll re-run the Ireland clam DNA with all the primer sets and see how it turns out. In the meantime, I’ll also order new universal primer sets to replace the existing, non-sterile sets.