PCR – pCR2.1/OsHV-1_ORF117 Colony Screens

Screened five colonies from yesterday’s transformation via PCR using M13 primers.

I don’t have any sequence for the actual insert, so am relying on assessing empty vector vs vector with insert, based on PCR amplicon size.

Master mix calcs:

2x GoTaq Green Master Mix: 80uL
M13 forward: 4uL
M13 reverse: 4uL
H2O: 88uL

Added 20uL to each PCR tube.

Colonies were selected randomly, streaked on a new LB Amp100 plate with a sterile pipet tip, and then added to the PCR tube.

Cycling params:

1 cycle

95C – 10mins

30 cycles:

95C – 15s
55C – 15s
72C – 30s

1 cycle

72C – 5mins

PCR reactions were run on a 1% agarose 1xTBE gel + EtBr.

5uL of O’GeneRuler DNA Ladder Mix was loaded for sizing.

Results:


 

 

 

 

 

 

 

Well, these results are confusing. Immediate conclusion is that all colonies screened are empty, due to the small size of the amplicons produced (<100bp). However, looking at a vector map of pCR2.1 (the vector that the OsHV-1 ORF117 is supposedly cloned in), there are ~200bp between the M13 forward and M13 reverse primers. So, even an empty vector should produce an amplicon larger than what is seen on this gel.

I’ll contact Tim Green to see if he can provide any insight (and/or any actual sequence for OsHV-1 ORF117 so that I can order an insert specific primer to aid in confirmation).

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.

PCR – Withering Syndrome cPCR Assay Limit of Detection Troubleshooting

Ran PCRs using the 3e7 copies of the p16RK7 plasmid curve used in the qPCR WS validation with some different Taq polymerases to see if the polymerase is the source of the

Results:

All samples were run in duplicate, except the GoTaq 1 Green Buff, where I accidentally loaded the 3e7 template into three of the four wells.