Since I’ve had no success in amplifying any of the Ireland Clam RLO (S/6/14 #19) DNA, I’m testing all the universal primer sets I’ve previously tried on the Ireland Clam DNA with red abalone DNA known to have heavy withering syndrome infection (confirmed via histology and qPCR) to verify that these universal primer sets actually work. I’m also using the withering syndrome primer sets on this DNA to function as a positive control.
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: 20150204 – Ireland Clam Troubleshooting GoTaq Flexi
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
Nothing. Since there’s nothing, I didn’t bother labelling the gel. So, this suggests that the PCR reactions aren’t working. Will get newer reagents to replace the 5yr+ old reagents I have been using. Also will try a different thermal cycler, just to rule out all possibilities.