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.
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)?
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.