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.