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.

qPCR – Old Curves, New Primer/Probe Test

Since the last run from earlier today failed using the WS3 curve (from 20140106), I’m repeating the run with two old curves from 20120731 (p16RK7 and p18RK7), since it seems very unlikely that all these brand new reagents would not work; it has to be the curve, right?

I did not use the SPUD assay.

Master mix calcs are here:

See the qPCR Report in the Results for plate layout/cycling params/etc.

All samples were run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-04-18 15-18-26_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-04-18 15-18-26_CC009827.pcrd

NOTE: Baseline threshold was NOT set at 400 RFUs, like we normally do since these qPCRs used two different polymerases which will have unique behavior compared to the old reagent (Bioline Immomix), as well as each other.

ABI Master Mix with p18RK7 Curve

ABI Master Mix with p16RK7 Curve

Promega Master Mix with p16RK7 Curve

Promega Master Mix with p18RK7 Curve

Both master mixes work, but it looks like Promega provides slightly better sensitivity (i.e. limit of detection) on both curves. Additionally, the p18RK7 curve may be very slightly better than the p16RK7 curve. Will continue using p16RK7 curve, as it was originally the curve we had been using before everything went to hell. However, there is not much left of the p16RK7 curve, so we may be switching to the p18RK7 curve very soon anyway.

qPCR – New Probe/Primer Test

Recently got new primer/probe set from ABI for the abalone withering syndrome qPCR assay. Testing out using 2x TaqMan Universal Master Mix (No UDG; ABI) and 2x GoTaq Probe Master Mix (Promega).

Used the WS3 standard curve (from 20140106).

Also ran the SPUD assay as a positive control.

Master mix calcs are here: 20140418 – qPCR WSN1 SPUD

See the qPCR Report in the Results for plate layout/cycling params/etc.

All samples were run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-04-18 10-59-00_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-04-18 10-59-00_CC009827.pcrd

The results are not good. Curve spacing is still jacked up and the SPUD assay is exhibiting behavior as though it is inhibited by amplification of WSN1! I’m going to test out these new reagents on a couple of the “old” curves, without the SPUD assay, and see what happens.

qPCR – ABI TaqMan Universal Master Mix (No UDG) Test

Ran qPCR using ABI’s TaqMan Universal Master Mix (No UDG) to test it out and see if we can get it to work. Used probe aliquot from 20140403.

Used p16RK7 curve (from 20120731), since it actually worked using SYBR green recently (see 20140324).

Master mix calcs are here: 20140409 – qPCR WSN1 ABI TaqMan Universal Master Mix

All samples were run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-04-09 16-21-53_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-04-09 16-21-53_CC009827.pcrd

Seeing the exact same results as the last two master mix tests (BioRad, Promega), in that the only sample of the curve to amplify is the 3e7 copy number. Granted, the replicates of this sample look TERRIBLE, but no other points in the dilution series light up at all! All three of these master mixes producing virtually the same result highly suggests that the primers and/or probe are faulty.

We have ordered a new probe from ABI. We will compare the two probes once it arrives.

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.

qPCR – Promega 2x GoTaq Probe Master Mix WSN1 Curve Comparisons

Trying out a new qPCR master mix for the WSN1 qPCR assay. Will compare the performance with two curves:

Neither curve amplified properly (only the highest copy number from each curve amplified) when used most recently with the BioRad Sso Advanced Probe Supermix (see 20140326).

Made (and used) fresh 10uM working stocks from the probe rec’d 10/1/2013.

Also, decided to set up a set using twice the amount of probe. This is just out of a weird fear that we’ve been reconstituting them incorrectly, so just trying something new. Really, just grasping at straws…

Master mix calcs are here: 20140403 – qPCR WSN1 Promega GoTaq Probe

See qPCR Report in the Results below for plate layout, cycling params, etc.

Results:

qPCR Report (PDF): Sam_2014-04-03 13-16-41_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-04-03 13-16-41_CC009827.pcrd

Only the two highest copy number from each curve amplified. This is exactly what I saw 20140326 when using the BioRad Sso Advanced probe Supermix!

It’s extremely difficult to even figure out what could be going wrong here. Why is there ANY amplification if this isn’t working??!! I would expect inhibited amplification or no amplification, if reagents were bad or something. This just continues to baffle.

Currently, the only thing that seems to work properly is using a SYBR green (Sso Fast EVAgreen; see 20140324) based assay. Knowing that, it seems to suggest that the probe that we have must be faulty. However, I still am not sure how there could be amplification in certain samples, but not in others…

qPCR – BioRad Sso Advanced Probe Supermix WSN1 Curve Comparisons

The run from Monday using the BioRad Sso Fast EvaGreen revealed that the old p16RK7 curve is useable. Want to see if probe has any effect.

Tested these three withering syndrome standard curves:

Additionally, ran three DNA samples, previously extracted from water filters that are known to be positive for withering syndrome via previous qPCRs run by Lisa:

  • TCA 0M A1
  • TCA +100M A1
  • TAF +100M A

Master mix calcs are here: 20140326 – qPCR WSN1 BioRad Probe

All samples run in duplicate.

See the qPCR Report in the Results below for plate layout, cycling params, etc.

Results:

qPCR Report (PDF):Sam_2014-03-26 16-16-43_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-03-26 16-16-43_CC009827.pcrd

Only the two highest copy number from each curve amplified. Will try the Promega 2x probe master mix next.

qPCR – BioRad SsoFast Supermix EvaGreen WSN1 Repeat

Due to the poor performance of the BioRad run on 20130321, this is repeating the run from 20140321, but using the “standard” WSN1 thermal profile, instead of the BioRad profile that was used on 20140321.

Again, tested three withering syndrome standard curves:

Master mix calcs are here: 20140321 – qPCR WSN1 BioRad EvaGreen

All samples run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-03-24 10-47-59_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-03-24 10-47-59_CC009827.pcrd

Well, changing the thermal profile made all the difference. The qPCR looks normal, in regards to amplification.

However, the WS3 curve still exhibits the weird spacing that has been seen by both Lisa and me previously.

The WS1 curve is bunched up at the two highest copy numbers as well as the two lowest copy numbers. But, the spacing on the rest of the curve looks good.

The p16RK7 curve looks pretty good. Although, the R^2 value isn’t as good as I’d expect, but it’s better than the other two curves.

Screenshots of each individual curve, along with it’s corresponding regression line and R^2 value are below for convenience.

Will repeat this PCR using the BioRad Sso Advanced Probe Supermix sample we have, using this thermal profile. Hopefully this will give us an idea of whether or not the probe has any impact. After that, regardless of the result, I feel fairly confident that we can just return to using the p16RK7 curve from 20120731 as our standard curve.

WS3 Curve

WS1 Curve

p16RK7 Curve

qPCR – BioRad SsoFast Supermix EvaGreen WSN1 Test

Ran qPCR testing the withering syndrome qPCR assay, but without the probe; just used the “sybr”-based BioRad SsoFast EvaGreen.

Tested three withering syndrome standard curves:

  • WS3
  • WS1
  • p16RK7 (from 20120731)

Master mix calcs are here: 20140321 – qPCR WSN1 BioRad EvaGreen

All samples run in duplicate.

See the qPCR Report in the Results below for plate layout, cycling params, etc.

Results:

qPCR Report (PDF): Sam_2014-03-21 14-15-53_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-03-21 14-15-53_CC009827.pcrd

Well, this worked terribly. Going to repeat, but extend the the denaturation and anneal/extension times. Not sure why the BioRad recommended times didn’t work. On a good note, the melt curves looked great.