qPCR – BioRad Sso Advanced Universal Probe Mix Test

Re-ran the qPCR from 20140306 to decrease activation time AND perform qPCR on additional curves. Changed the following in the thermal profile:

Activation Time: Was 10mins, Changed to 30s (30s recommended in BioRad product information sheet for SsoAdvanced Universal Probes Supermix).

Anneal/Extension Time: Was 1min, Changed to 30s (30s recommended in BioRad product information sheet for SsoAdvanced Universal Probes Supermix).

Curves used were:

  • WS3
  • WS1
  • p16RK7 (from 20120731)

Hematodinium Curve A (3e6 – 30 copies)

Master mix calcs are here: 20140311 – qPCR WSN1 SPUD BioRad

All samples run in duplicate.

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

Results:

qPCR Report (PDF): Sam_2014-03-11 15-49-37_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-03-11 15-49-37_CC009827.pcrd

Well, that failed miserably in all facets! Almost no amplification of any of the standard curves (only 2 out of 30 showed any amplification) and the SPUD internal amplification controls were all over the place.

I guess we should lengthen the activation time. Grrrr.

Ha! Just realized that OF COURSE the hematodinium failed! Didn’t use hemat primers! Duh!

qPCR – BioRad Sso Advanced Universal Probe Mix Test

Ran qPCR on the WS3 curve with the withering syndrome qPCR assay and using the SPUD internal amplification control (1:10 million dilution as template) assay.

Master mix calcs are here: 20140306 – qPCR WSN1 SPUD BioRad

All samples were run in duplicate.

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

Results:

qPCR Report (PDF):
qPCR Data File (CFX96):

Quick summary, the standard curve data looked virtually identical to Lisa’s run from 3/5/2014, which is not good. Curve dilutions are 10-fold, however the spacing of the curve doesn’t exhibit the expected difference in Cq of 3.32 per 10-fold dilution.

The SPUD assay was very consistent and all samples came up at virtually the same time.

After discussing with Lisa, we realized that the activation time that we were using on the Thermal Profile was far too long for this polymerase. Will try re-running and decreasing the activation time. Will also perform qPCR on the other curves.

qPCR – Immomix Vial Checks

Ran qPCR on a set of previously unopened box of Immomix (lot #112E) to verify that all vials in the box are good, due to the variability we have experienced within lot numbers in our most recent troubleshooting of the withering syndrome qPCR assay.

The nine vials in the box were numbered one through nine and then individual master mixes were made from each with the SPUD internal amplification control (1:10 million dilution as template) assay.

All samples were run in triplicate.

Master mix calcs are here: 20140124 – qPCR Immomix Checks

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

Results:

qPCR Report (PDF): Sam_2014-01-24 15-33-53_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-01-24 15-33-53_CC009827.pcrd

Vial 5 is the ONLY vial that performed as expected! A single vial out of 10 vials! Vial 3 is potentially useable.

All other vials showed very little amplification, as well as variation in their amplification. Performance was absolutely dreadful. Considering these were reactions where the master mixes were simply distributed into wells, the variation seen in some of them is baffling.

Will contact Bioline about lot number expiration dates, as well as getting reimbursed (in Immomix) for all the time/money we’ve wasted troubleshooting.

qPCR – Withering Syndrome Standard Curves

Ran qPCR on one of the new curves (WS-3; from 20140106) using a fresh vial of Immomix and Lisa’s current vial of BSA. Ran both WSN1 and SPUD assay (1:10 million dilution of template). Used Sigma water.

All samples were run in duplicate.

Master mix calcs are here: 20140115 – qPCR WSN1 SPUD New Curves

See the qPCR Report for plate layout, cycling params etc. Baseline threshold was set to 400 RFUs.

Results:

qPCR Report (PDF): Sam_2014-01-15 14-51-24_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-01-15 14-51-24_CC009827.pcrd

 

IT WORKED!!!!!!! Standard curve looks perfect, SPUD looks perfect; yes!!! Finally! The withering syndrome qPCR assay is back in effect!!!

Except, this begs the question of what the source of the problem is/was. I suspect it is a problem with the Immomix, not the BSA.

qPCR – SPUD Assay Volume and Reagents Tests

Ran qPCRs comparing Lisa’s reagent aliquots (Immomix and BSA) to mine, as well as the difference in reaction volumes. Used 1:10 million dilution SPUD template and Sigma water.

Master mix calcs are here: 20140115 – qPCR SPUD Volume and Reagents Test

All samples were run in triplicate. See qPCR Report below for plate layout, cycling params, etc.

Results:

qPCR Report (PDF): Sam_2014-01-15 11-23-32_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-01-15 11-23-32_CC009827.pcrd

Not surprisingly, the result concludes that I have bad aliquots of reagents, despite the fact that both the Immomix and the BSA are of the same lot #s as Lisa!

Will test out my new curves with fresh aliquots of both Immomix and BSA.

qPCR – SPUD Assay Template and Water Comparison-2

Repeated the assay done earlier today, but changed the reaction volume to 21uL to mirror what Lisa did yesterday when it worked. This is odd, because she final concentration of the 2x Immomix does not end up being 1x (based on her master mix volumes).

I also used the same reagents (Immomix, BSA) that Lisa used in her reactions yesterday.

Master mix calcs are here: 20140114 – qPCR SPUD Template Comparison-2

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

Results:

qPCR Report (PDF): Sam_2014-01-14 16-53-13_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-01-14 16-53-13_CC009827.pcrd

The results are great! All samples amplify AND there is absolutely no water effect.

But, what does this mean?

  • Need to run 21uL reactions?

  • My reagents are bad (Immomix, BSA)?

  • Both?

It shouldn’t be my reagents, as both Lisa and my Immomix are the same lot # and were from the same box. I’ve used two different aliquots of BSA in the last couple of days, so that shouldn’t be an issue, either.

BUT!!!! I just realised that Lisa’s 21uL reactions don’t change the amount of each component used (we’re each using the same volume of each reagent per reaction), just the final volume. That means the final concentrations of ALL components are different than what they are in 25uL reaction.

qPCR – SPUD Assay Template and Water Comparison

Ran qPCR with just the SPUD assay, using all three available template dilutions:

  • 1:10,000
  • 1:100,000
  • 1:10 million

Also set up one set using the PCR water in the hood (from Friedman Lab Nanopure 10/9/2013) and one set with Sigma PCR water.

Master mix calcs are here: 20140114 – qPCR SPUD Template Comparison.

All samples were run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-01-14 11-07-32_CC009827.pdf
qPCR Data File (CFX): Sam_2014-01-14 11-07-32_CC009827.pcrd

The only amplification was in the 1:10,000,000 (10e-7) with Sigma PCR water. However, only one of the two replicates amplified! I’m not even sure how this is possible since the master mix contains all elements of the reaction. Additionally, the amplification came up at cycle 37, which is about ~3-4 cycles later than what Lisa’s reactions came up at. The reactions were simply distributed to the wells on the plates and then run. I checked the wells after qPCR to verify that all of them had liquid.

Will re-run to get this right. I will also change the reaction volume to 21uL (which is the same as Lisa’s, which worked perfectly), despite the fact that final volume does not result in a 1x final concentration of the polymerase.

qPCR – SUPD Assay with Different Polymerase

Since the water source tests from 20140109 showed that water source had no effect AND the SPUD assay only amplified in wells that showed WSN1 amplification, I decided to try a different polymerase that I know is functional and performs well. I used 2x Sso Fast EvaGreen (BioRad) from the Roberts Lab instead of 2x Immomix (Bioline) from the Friedman Lab. Since the Sso Fast EvaGreen already contains a fluorescent SYBR analog, I did NOT use the RLP probe that is normally used in the WSN1 qPCR.

For template DNA, I used the serial dilution set from 20140109 of undigested pCR2.1/WS-3 in Roberts Lab Nanopure water (from 7/11/2012). Additionally, I ran a no template control with water and a no template control with no template added at all.

Master mix calcs are here (20140113 – WS qPCR SPUD SsoFast Test). See the Results below for plate layout, cycling params, etc. Baseline threshold was set at 400 RFUs for both fluorophores. All samples were run in duplicate (except the empty no template control; only a single well was run).

Results:

qPCR Report (PDF): Sam_2014-01-13 10-21-02_CC009827.pdf
qPCR Data File (CFX): Sam_2014-01-13 10-21-02_CC009827.pcrd

The results are interesting. Most importantly, the SPUD assay amplifies in all samples, including the negative controls. But, there is WSN1 amplification in all of the negative controls! Additionally, all of the WSN1 samples also amplify and exhibit the proper spacing between Cq for each 10-fold dilution (3.32 Cqs) for almost all of the samples. However, the SPUD assay still shows an equidistant shift in Cq when there’s a shift in WSN1 Cq. Also, it might be worth noting that the amplification levels (i.e. the raw fluorescent units) are way, way, way higher than what is normally produced using Immomix and the WSN1 probe.

This is too crazy. Lisa is going to try to run her old Hematodinium curve with the SPUD assay to see what happens.

UPDATE 1/14/2014

Lisa ran her samples with three different polymerases (2x BioRad Sso Fast Probe, 2x SensiMix, and 2x Immomix). Her curves all look perfect and the SPUD assay amplified equally in all samples, including the WSN1 negative controls. Interestingly, the samples run with BioRad showed a general shift of ~3 Cqs EARLIER than the corresponding samples using the other two polymerases. This effect is also seen in the SPUD assay which comes up at ~30 Cq in the BioRad samples, but comes up at ~33 Cq in the other two polymerases. However, the BioRad exhibits some strange amplification patterns at the lower end of the standard curve that is not present in with the other two polymerases.

Lisa used different SPUD assay conditions than what I’ve been using. She used 0.3uL of the 1:10,000,000 per reaction. I have been using 2uL of the 1:10,000 template per reaction. Is it possible that my reactions are suffering inhibition due to molecular crowding (i.e. so much DNA in the reaction that the DNA is inhibiting the reaction)? Will test this out. Additionally, will have Lisa try her hand at running the newest WS standard curves to see if she has any luck.

Lisa also used Sigma PCR H2O instead of the Nanopure H2O.

Lisa also used a reaction volume of 21uL. This is odd, because it does not result in a 1x final concentration of the polymerases (she used 12.5uL of 2x polymerase mix in each reaction).

Lisa used different cycling parameters for run, which was for the Hematodinium curves (95C -15s, 60C – 30s, 72C 30s).

qPCR – SPUD Assay with Different Water Templates

The results from yesterday’s qPCR suggest that the water being used to make lab solutions and used in our PCRs may be bad. So, I tested out three different sources of molecular grade water (18 megaohms).

Also prepared five serial, 10-fold dilutions (for each of the two plasmids (pCR2.1/WS-1, and WS-3) that were isolated on 12/11/2013. Each of these were diluted in three different sources of water:

  • old – Friedman Lab Nanopure (from 10/9/2013)
  • new – Friedman Lab Nanopure (from 1/9/2014)
  • RL – Roberts Lab Nanopure (7/11/2012)

Additionally, I used each of these water sources for no template control samples. Finally, I also left wells with only 23uL of the qPCR master mix and did not add any template of any kind.

Master mix calcs are here (20140109 – WS qPCR SPUD Waters Test). See the Results below for plate layout, cycling params, etc. Baseline threshold was set at 400 RFUs for both fluorophores. All samples were run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-01-09 15-33-48_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-01-09 15-33-48_CC009827.pcrd

  • Water source does not seem to have any effect

  • SPUD amplification only occurs in wells where there is WSN1 amplification.

  • SPUD Cq is dependent upon WSN1 Cq (i.e. when WSN1 amplification comes up at a later Cq, SPUD amplification also comes up at a later Cq in the same well)

  • 10-fold dilutions are not reflected in data; expectation is a 3.32 difference in Cq for each 10-fold dilution. This problem is what we started seeing when the previously functional curves began failing

The next things I will try are:

  • Use store-bought PCR grade water. Discussing the issue with Brent, he suggested that maybe the pH of the buildings’ water supplies is too low (he has experienced in issue with this previously in his tenure at the university). He also suggested we review our data for all of this troubleshooting and see if there’s a seasonal effect, which could be caused by the city of Seattle switching different source watersheds for their water supply during different seasons.

  • Try SYBR-based qPCR using WSN1 primers, but no probe. Will still use SPUD in these reactions.

qPCR – SPUD Assay

As part of the withering syndrome qPCR assay troubleshooting, I ran the SPUD assay by itself and within the standard curves.

Master mix calcs are here (20140108 – WS qPCR SPUD Test).

Ran the SPUD assay with the two NcoI linearized plasmid stocks (WS1 and WS3), and with the WS1 standard curve. Additionally, ran the SPUD assay on its own, using a dilution series of the SPUD template DNA (which already exists as a 1:10,000 dilution of the stock DNA) in the same Low TE (made 10/9/2013 SJW) that was used for the WS standard curves. See the Results below for plate layout, cycling params, etc. All samples were run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-01-08 14-12-27_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-01-08 14-12-27_CC009827.pcrd