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.

PCR – Ireland Clam DNA 18s

Since the previous PCR didn’t amplify anything using four different universal 16s (prokaryote) primers, I am testing to verify that the two extractions (QIAamp Fast DNA Stool Mini Kit & the DNeasy Kit; Qiagen) actually isolated any amplifiable DNA using universal 18s (eukaryote) primers.

First, quantified the samples to verify that DNA actually exists in these two samples:

20150128_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

Sample Concentration (ng/uL)
Clam DNeasy Kit 4.432
Clam Stool Kit 6.184

The yields are surprisingly low, particularly for the DNeasy Kit sample.  In a total elution volume of 200uL, that means I only extracted 800ng…

Due to low DNA concentrations, I used 10uL of each sample in the PCRs.

Master mix calcs are here: 20150129 – cPCR Clam Universal 18s

Samples were run in duplicate.

Cycling params:

  • 1 cycle of 10mins
  • 40 cycles of:
    95C – 15s
    50C – 15s
    72C – 2mins

Results:

Ladder used was Hyperladder I (Bioline).

Neither sample produced any amplification.  The blurry “bands” that correspond to ~100bp are likely RNA carryover from the DNA isolation procedure.  They are not the amplicon we are looking for.  Additionally, they are not primer dimers, as these “bands” do not appear in the NTC.

I believe there is a small quantity of tissue debris in the original EtOH sample tube.  I will attempt to isolate some DNA from this debris and will repeat both the 16s and 18s PCRs.

Ligation/Transformation – Elene’s 18s MLO PCR Product & Transfomation of WS-Phage Ligation (from 20121206)

Ligated Elene’s purified 18s MLO PCR product from 11/26/2012 into pCR2.1 using the TOPO TA Kit (Invitrogen). Since there are likely to be multiple species of 18s sequence, I performed a full reaction and incubated ligation at R/T for 30mins (instead of 5mins) to maximize the number of transformants.

Both transformations were handled according to the Invitrogen TOPO TA Cloning manual, utilizing chemically competent TOP10 cells (Invitrogen).

Results:

Elene’s 18s MLO – Got a small number of potential transformants (~15). Will check transformants via colony PCR and will re-streak on a fresh plate.

WS-Phage – Got no transformants at all; not even blue colonies (negatives). This would suggest that the ligation reaction failed. Possibly due to the age of the T4 DNA Ligase that was used. Will get new ligase and attempt ligation/transformation again.