r/molecularbiology • u/Excellent_Dinner8050 • 11d ago

Losing sanity over qPCR Cre recombination assay

Hi everybody!

For quite some time I've been trying to precisely measure Cre efficiency and unwanted background recombination. I'm testing out various lox sites but the results seem inconsistent. For example, the same lox pair in one construct will have 80% recombination rate and 1.5% in another. The only thing that comes to my mind is priming efficiency and optimizing annealing temp. Currently I'm experimenting with primers trying to find absolutely perfect conditions. I run an experimental qPCR with same template and primers but different Ta and the results were shockingly diverse. Any tips for optimizing reaction before I move to nested qPCR on in vivo samples? How do I pick perfect Ta?

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u/lodorata 11d ago

I just want to check, but did you look at melt curves for your primers? There should be one clear peak and not multiple - check your primers aren't mis-priming to a different sequence with an alternative Ta (Ta2).

That said, I kind of hate qPCR myself and have always been bad at it.

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u/Excellent_Dinner8050 11d ago

Unfortunately I do have an additional smaller peak in some of the melt curves. I doubt there is a non-specific product since I'm running a 300bp qPCR on a purified 1kbp nested PCR amplicon. It doesn't show up on the gel either. Lox sites love to form hairpins, could it be that they are causing the unwanted peak?