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 – 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 – Withering Syndrome Standard Curves

Ran qPCR on two new WS standard curves using the dilutions set up earlier today. Also ran undiluted digests of both plasmids as potential positive controls.

Master mix calcs are here (20140106 – qPCR RLO New Curves). See the Results below for plate layout, cycling params, etc. All samples were run in duplicate.

 

Results:

qPCR Report (PDF): Sam_2014-01-06 15-49-04_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-01-06 15-49-04_CC009827.pcrd

No amplification of any kind in any of the two standard curves! However, there was amplification of both of the undiluted digest samples!

qPCR – New Withering Syndrome Plasmid Curve (from 20131106)

Ran qPCR on new p16RK7 plasmid curve.

Master mix calcs are here: 20131119 – qPCR RLO New Curves

Results:

qPCR Report (PDF): Sam_2013-11-19 11-56-58_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-11-19 11-56-58_CC009827.pcrd

No amplification of either curve! This sucks. Will brainstorm additional troubleshooting ideas…

qPCR – Abalone RLO qPCR Assay

Testing out new stocks of primers and probe received this week. Stocks (and working stocks) will be made with different batches of low TE and PCR water than what we have been using in the hood. Will use the RLO p16RK7 plasmid curve from 20120731. Master mix calcs are here.

Results:

qPCR Report (PDF): Sam_2013-10-09 13-33-42_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-10-09 13-33-42_CC009827.pcrd

qPCR – Abalone RLO qPCR assay; trying “new” reagent aliquots and additional plasmid curve

Since yesterday’s comparison of thermal cyclers yielded identical results, it’s clear that the machine is not the issue. I’m repeating yesterday’s qPCR, but using the aliquots of reagents from Lisa that I used on DATE. Additionally, I’m checking a fresh, separate dilution of the RLO plasmid curve (p16RK7 from 20120731) that Lisa made last week. Master mix is the same as yesterday.

Results:

qPCR Report (PDF): Sam_2013-09-25 10-52-18_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-09-25 10-52-18_CC009827.pcrd