PCR – Ireland Clam RLO DNA S/6/14 #19 (from 20150130)

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:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 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.

Results:

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).

PCR – Universal Primers w/New Master Mix

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:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 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

Results:

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.

PCR – Test Universal Primers with Abalone DNA

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:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 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

Results:

 

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.

DNA Quantification & PCR – Ireland Clam S/6/14 #19 DNA

Quantification

Quantified the DNA isolated 20150130 via NanoDrop1000 (Thermo Fisher) for quick assessment of DNA.

Although the NanoDrop1000 overestimates actual yields, this is still interesting because the overall yield of this sample is greater than either of the samples isolated on 20150122, yet had significantly less starting material.

 

PCR

Ran PCRs with the following “universal” primer sets in attempt to amplify a 16s (prokaryote) fragment from the RLO that is present in this sample. Additionally, ran a universal 18s (eukaryote) primer set to verify the presence of any amplifiable DNA in the sample, in case none of the 16s primers work.

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 18s EUK 581 F, 18s EUK 1134 R

Master mix calcs are here: 20150203 – cPCR Clam debris DNeasy

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 from yesterday’s PCR out on a 0.8% agarose,  1x TBE gel w/EtBr

Results:

Ladder is Hyperladder I (Bioline)

Well, ironically, the only thing that shows amplification is the no template controls (NTC) in the universal 16s primer set!  The only useful aspect of this is that it demonstrates that the reagents are functional.

The universal 18s primers don’t seem to amplify anything, either.

Tomorrow, I’ll test these primers out on DNA that I know will amplify, instead of these new clam DNA samples.