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 – Universal 16s in Ireland Clam DNA

In an attempt to identify a potential rickettsia-like organism (RLO) in the clam sample, ran PCRs with various universal 16s primers:

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

Template DNA used were the Ireland clam DNA isolated yesterday/today with the Qiagen stool/DNeasy kits, respectively.

Master mix calcs are here: 20150122 – cPCR 20150122 – cPCR Clam Universal 16s

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, low 1x TAE gel w/EtBr

Results:

No amplification in either sample (stool or DNeasy kit DNA) with any of the four primer sets.  Will discuss with Carolyn how she wants to proceed.


 

PCR – In-situ Hybridization (ISH) Probe

Ran probe-labeling PCRs to use in in-situ hybridization (ISH). Generated PCR probes for the following:

Phage ORF20

Phage ORF25

Withering Syndrome (p18RK7, 3e6)

PCR calculations are here: 20141008 – ISH Probe 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.

Cycling Params:

  1. 95C – 5mins

  2. 95C – 15s

  3. 55C – 15s

  4. 72C – 30s

  5. Go to Step 2, repeat 39 times.

  6. 72C – 10mins

Ran 5uL of each sample on a 1% agarose 1x TBE gel, stained with EtBr.

Results:

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.

Probes were stored @ -20C in my -20C box.

PCR – Withering Syndrome Bacteriophage Clone Screen

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.

Master mix calcs are here: 20140909 – Phage Colony Screens

Cycling params:

1 cycle:

  • 95C – 10mins

40 cycles:

  • 95C – 15s
  • 55C – 15s
  • 72C – 30s

Results:

Top half of gel are the pCR2.1/ORF20 colonies.

Bottom half of gel are the pCR2.1/ORF25 colonies.

All amplified. Will select one of each from the gridded plates for plasmid isolation for use in qPCR standard curves and in-situ hybridization (ISH).

PCR – Withering Syndrome Phage

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:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Master mix calcs are here: 201400813 – PCR WS phage

Cycling params:

Ran samples on 1.2% 1x TBE + EtBr.

Results:

Ladder: O’GeneRuler 100bp DNA Ladder (ThermoFisher)

Good amplification from all three primer sets. The pinto abalone sample (UW08:22-65) that should be naive for withering syndrome and phage did not amplify as expected.

Excised bands from each primer set in the 06:6-41 group and purified using Ultrafree DA spin columns (Millipore). Will save for potential cloning usage, depending on future results.

PCR – Universal Phage Primers Test

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.

Results:

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.

PCR – Universal Phage Primers Test

Used universal phage primers on Ab Endo 2010 tissue sample 10:13-60. Sample was identified as having the highest level (2.5) of “PE-VAR” of all extracted samples to-date.

Master mix calcs are here: 20140404 – cPCR Universal Phage

Cycling params are as follows:

Primers: g231 & g232 (UPIDs: 80 & 81)

1 cycle:

  • 95C – 5m

37 cycles:

  • 95C – 45s
  • 50C – 1m
  • 72C – 45s

1 cycle:

  • 72C – 10m

Primers: podoF, podoR1, podoR2 (UPIDs: 84, 83, 82)

1 cycle:

  • 95C – 5m

6 cycles:

  • 95C – 30s
  • 50C – 47C (0.5C/cycle) – 30s
  • 72C – 1m

34 cycles:

  • 95C – 30s
  • 47C – 30s
  • 72C – 1m

1 cycle:

  • 72C – 10m

Results:

No amplification in any samples.  No gel image taken.  Will repeat tomorrow with increased quantity of template DNA.

PCR – Colony Screens from Withering Syndrome 16s Cloning from yesterday

Selected 10 white colonies for PCR and restreaking. Master mix calcs are here.

Results:

 

Ladder = Hyperladder I (Bioline)

All colonies produced a band of the expected size (~1500bp). Will select three colonies to grow up for plasmid isolation.

PCR – Full Length AFF133090 (Abalone Withering Syndrome 16s)

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:

  • p16RK7 (from 20120718)
  • p18RK7 (from 20120718)
  • pWC8 (from 20120718)
  • p16RK7 A (from 20131106)

Primers used:

  • WS_16s_1_F
  • WS_16s_1501_R

Master mix calcs are here: 20131203 – cPCR WS Full Length

All reactions were run in duplicate.

Cycling params:

  • 95C – 10m

40 cycles of:

  • 95C – 15s
  • 55C – 15s
  • 72C – 2m

Results:

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.