PCR – Universal Primers w/New Master Mix

Since the previous check of the various universal primers with abalone DNA (sample 09:8-20) failed to amplify, even with withering syndrome primers, I’m testing repeating that PCR using a newer/different PCR master mix.

Template DNA is: 09:20-08 (from tissue)

Background info for template DNA is here: Red/Pink/Pinto

Primers being used are:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 18s EUK 581 F, 18s EUK 1134 R
  • WSN1 (withering syndrome)

Master mix calcs are here: 20150212 – cPCR Universal Primers 09:8-20 Apex Red MM

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 out on a 0.8% agarose,  1x TBE gel w/EtBr

Results:

Well, this is a good result.  It demonstrates that the previous reagents that I had been using are no good. The primers work.  However, it does appear that all of the universal primers (excluding the 18s and EHR) are contaminated.  All of these primer sets were stocks that were prepared by other people and none of them were marked as being sterile (which they should be).  Regardless, I’ll re-run the Ireland clam DNA with all the primer sets and see how it turns out.  In the meantime, I’ll also order new universal primer sets to replace the existing, non-sterile sets.

PCR – Test Universal Primers with Abalone DNA

Since I’ve had no success in amplifying any of the Ireland Clam RLO (S/6/14 #19) DNA, I’m testing all the universal primer sets I’ve previously tried on the Ireland Clam DNA with red abalone DNA known to have heavy withering syndrome infection (confirmed via histology and qPCR) to verify that these universal primer sets actually work.  I’m also using the withering syndrome primer sets on this DNA to function as a positive control.

Template DNA is: 09:20-08 (from tissue)

Background info for template DNA is here: Red/Pink/Pinto

Primers being used are:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 18s EUK 581 F, 18s EUK 1134 R
  • WSN1 (withering syndrome)

Master mix calcs are here: 20150204 – Ireland Clam Troubleshooting GoTaq Flexi

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 out on a 0.8% agarose,  1x TBE gel w/EtBr

Results:

 

Nothing.  Since there’s nothing, I didn’t bother labelling the gel. So, this suggests that the PCR reactions aren’t working.  Will get newer reagents to replace the 5yr+ old reagents I have been using.  Also will try a different thermal cycler, just to rule out all possibilities.

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.

Plate Reader – Tecan Plate Reader Problem

The Tecan plate reader is behaving strangely. I tried to quantify plasmid DNA samples isolated on Friday, but this is how the plate turned out:

The areas NOT demarcated by the black lines should all appear as dark blue (i.e. no fluorescence detected). I’ve never seen a plate look like this before.

To be sure that the issue wasn’t specific to just the plate I was using, I ran an empty plate through the same Tecan method.

Here’s the pic of an entirely empty/blank plate (i.e. no samples loaded in any wells; just an empty, dry plate):

Empty Plate

Ideally, these should all be the same exact color (dark blue), since there should be no fluorescence in any wells.

I probably need to contact Tecan regarding this issue…

qPCR – Withering Syndrome Assay with New IDT Primers

It dawned on me that the qPCRs I ran on 20140909 comparing different stocks of primers, using the new IDT probe (which all ended up looking bad), all used primer stocks that had been reconstituted with low TE buffer made in the lab, as opposed to “store bought.” Could this actually be the reason that the IDT probes (which come lyophilized and require reconstitution prior to use) don’t seem to work, while the ABI probe (which comes as a liquid, 100uM stock) does?

Received new IDT primer stocks of WSN1F/R and reconstituted them with the store bought low TE buffer from IDT.

All samples were run in duplicate. See the qPCR Report (in Results below) for plate layout, cycling params, etc.

Baseline Threshold set to 580 RFUs, as previously determined by Lisa for use with the Promega master mix.

Results:

qPCR Report (PDF): Sam_2014-09-19 10-55-33_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-09-19 10-55-33_CC009827.pcrd

I’ll be damned! Our lab-made low TE buffer seems to have been the culprit! Both IDT and ABI probes performed perfectly and are virtually identical. See the amplification plots and standard curve plots below for each of the two probes. Both exhibit R^2 values of 0.999.

ABI Probe Amplificaton

ABI Probe Standard Curve

IDT Probe Amplification

IDT Probe Standard Curve

qPCR – Withering Syndrome Probes Comparison

Due to poor performance seen in yesterday’s second run, which used the IDT probe instead of the ABI probe, I will run the p18RK7 curve (from 20120731) with both the IDT and the ABI RLP_p probes to see if the probe is the underlying issue.

Master mix calcs are here: 20140827 – qPCR IDT-ABI Probe Comparison

All samples were run in duplicate.

Plate layout, cycling params, etc can be seen in the qPCR Report (see Results below).

Baseline threshold set to 580 RFUs.

Results:

qPCR Report (PDF): Sam_2014-08-27 10-32-19_CC009827.pdf

qPCR Date File (CFX96): Sam_2014-08-27 10-32-19_CC009827.pcrd

Quick summary: IDT probe is bad, ABI probe is good.

However, I’ve just realized that there might be more to this than just a bad probe from IDT. The amplification profile is suspiciously similar to what we were seeing with the bad probe(s) from Biosearch Technologies. The only thing that differentiates the IDT and Biosearch Technologies probes from the ABI probe is that the ABI probe already arrived reconstituted at 100uM. Biosearch Technologies and IDT probes arrive lyophilized and have to be reconstituted by us. Is it possible that the buffer we’re using is degrading the probes? Will order new IDT probe and order IDT’s version of low TE buffer (called IDTE).

Here are the amplification and standard curve plots:

IDT Amplification

IDT Standard Curve

ABI Amplification

ABI Standard Curve

qPCR – p18RK7 Curve IDT Primer Test

Quick withering syndrome qPCR assay test using the ABI RLP_p probe and IDT primers. This is to see if we can still use IDT primers that have NOT been ABI HPLC purified. This would be a significant cost savings, as HPLC purified primers from ABI cost ~$60 each (“regular” IDT primers only cost ~$6 each). This should work.

Master mix calcs are here: 20140502 – qPCR p18RK7 Curve WSN1 IDT Primers

Used p18RK7 curve from 20120730 because we are running low on the p16RK7 curve (from the same date).

Plate layout, cycling params, etc can be found in the qPCR Report in the Results below.

Results:

qPCR Report (PDF): Sam_2014-05-02 09-42-38_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-02 09-42-38_CC009827.pcrd

Not surprising,the IDT primers work just fine. Will continue to use/order IDT primers for withering syndrome PCRs.

qPCR – p16RK7 Curve Old Primers and Probe Test, Lisa Sample Check

Ran qPCRs testing the “old” primers/probe (IDT primers, Biosearch Technologies Probe) using ABI 2x qPCR master mix and Promega 2x qPCR master mix.

The curve used was the p16RK7 from 20120731. This was used because it worked perfectly on 20140418.

Also ran a qPCR with all ABI components along with the following set of samples that Lisa needed checked for positive/negative of withering syndrome:

2010 Water Filter DNAs (Site 8 extracted 20120228 and Site 3 extracted 20120229 by me):

  • SNI Site 8 0M Rep. 1
  • SNI Site 8 +100M Rep. 1
  • SNI Site 8 -100M #1
  • SNI Site 8 +500M #1
  • SNI Site 8 -500M #1
  • SNI Site 3 0M #1
  • SNI Site 3 +100M #1
  • SNI Site 3 -100M #1
  • SNI Site 3 +500M #1
  • SNI Site 3 -500M #1

Master mix calcs are here: 20140422 – qPCR p16RK7 Curve Old Probe

Plate layout, cycling params, etc. can be found in the qPCR Report in the Results below.

All samples were run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-04-22 15-50-08_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-04-22 15-50-08_CC009827.pcrd

NOTE: Since this used a different polymerase (ABI) than what has been used in the past for this assay (Bioline), I did NOT adjust the baseline threshold. A new baseline threshold will have to be decided upon.

The qPCR run with the new probe/primer set worked wonderfully! The qPCRs with the old primer/probes failed miserably!

Here are the amplification curves and standard curve graph from the new primer/probe sets using the ABI master mix. The brown colored curves are the standard curve. The blue colored curves are some of Lisa’s samples.

Below are the other TWO curves (yep, both of them!) run with the old primer/probe set. Purple colored curves are Promega master mix, green colored curves are ABI master mix.