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:
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.
Used the PCR DIG Probe Synthesis Kit (Roche), with 10pg of template for each probe. Ran a DIG positive and DIG negative sample for each sample.
95C – 5mins
95C – 15s
55C – 15s
72C – 30s
Go to Step 2, repeat 39 times.
72C – 10mins
Ran 5uL of each sample on a 1% agarose 1x TBE gel, stained with EtBr.
Ladder: Hyperladder I (Bioline)
ORF25 and p18RK7 PCRs seemed to have worked as expected. This is evidenced by larger molecular weight bands in the DIG-positive samples (the DIG molecules incorporated during PCR result in slower migration through the gel).
However, the ORF20 PCR doesn’t seemed to have worked in either the DIG-postiive nor the DIG-negative. I won’t bother re-running this, since the ORF25 will also function as a probe for detection of withering syndrome phage.
10 colonies from both ligations (pCR2.1/ORF20 & pCR2.1/ORF25) were picked with clean pipette tips, streaked on a different LB-Amp50+X-gal plate that had been gridded and numbered, and then used to inoculate the PCR reactions.
PCRs were performed with respective phage primers.
We received MiSeq data back from Stan Langevin (samples submitted 20140717) and he believes he has sequenced the entire WS phage. Carolyn and Colleen designed some primers on two of the open reading frames annotated by Stan. Ran PCR with the three primer sets to test out:
Repeated the PCR from 20140404, but used 2uL of the completed PCR reactions as template for a subsequent round of PCRs, in case the first round of PCR is insufficient to generate enough amplicon to be visible on a gel.
See 20140404 for all master mix calcs, initial template source, etc.
First and second round PCR reactions were run on a 1.0% agarose 1xTBE gel stained with EtBr.
Ladder used was Hyperladder I (Bioline)
No amplification in any samples, in either round of the PCRs. For kicks, will do a run with SYBR green for a more sensitive assessment of whether or not there’s any amplification occurring.
Ran conventional PCR to amplify the full length abalone withering syndrome 16s product for subsequent cloning. Used four different DNA plasmid preps as template, for comparison purposes. Plasmid preps used were:
Ran 20uL (of 50uL reaction) of each sample on 0.8% TBE gel.
Lanes (left ro right):
1 – Hyperladder I (Bioline)
2 – p16RK7 (from 20120718)
3 – p16RK7 (from 20120718)
4 – p18RK7 (from 20120718)
5 – p18RK7 (from 20120718)
6 – pWC8 (from 20120718)
7 – pWC8 (from 20120718)
8 – p16RK7 A (from 20131106)
9 – p16RK7 A (from 20131106)
10 – NTC
11 – NTC
Expected a band of ~1500bp. Bands of all samples (except pWC8) run at ~1500bp. pWC8 runs a bit higher and will not be used for cloning, as part of our troubleshooting the failure of our plasmid qPCR standard curve for withering syndrome.