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.

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