Hi all, it happened a very strange thing that never happened to me before.
I run a PCR with primers for genotyping zebrafish embryos after CRISPR-Cas9 injection. I tried changing different parameters, but always no results (gDNA was extracted from 24dpf embryos). Then I tried with gDNA extracted from adult fins, same identical PCR conditions, and the primers worked. I would have expected something like that if I'd used cDNA, but how could that be possible with gDNA?
Thank you in advance!
How are the primers constructed? Do they amplify the sequence with or without a successful CRISPR? Have you ever had the primers work on embryos? Were the adult fins from fish that had already had the CRISPR performed prior?
Hi Joshua and thank you for your answer. Maybe it is worth the mention that I've tried them also on control fish, both embryos and adults. So no CRISPR has been performed on these latter. That's how I realized that primers work on adults but not on embryos.
Primers are intended to amplify the region comprising the target exon of the CRISPR + the flanking intron, so the sequence without the successful CRISPR (whose efficiency would have been assessed through enzymatic digestion). They have been previously tested from a technician and unfortunately I don't have this information, but I assume they were tested in adults.
So, leaving aside for a moment the CRISPR thing, it's strange that "normal" primers don't work if used with embryonic gDNA, whereas they work on adult gDNA.
Thank you.
That's not supposed to happen. It might mean that the primers are working so poorly that they're sensitive to seemingly small variations in template quality.
According to what you told us, these differences could still be random. If you tested 5x embryos vs. 5x adults, each prepped independently on different days, would you still see the effect? Or would it just be random? No way to know until you try.
In the end it doesn't matter. You just don't want a primer that acts up like that. Try to optimize PCR conditions, or design new primers if at all possible.
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