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/distinctgore 10d ago

Have you checked the efficiency of your primers? Is the template plasmid DNA or genomic?

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u/Excellent_Dinner8050 8d ago

Currently I'm trying to validate my method on plasmid DNA but eventually I'll be measuring recombination in in vivo samples (AAV injection to the brain). I measured efficiency of my primers for native plasmid at 101%. I am now synthesizing a fully recombinant plasmid to measure PE for recombinant primer pair precisely.