Recently I performed an yeast transformation and I got a few colonies in the selective plate. The strain is very modified, with four auxotrophic markers (CRISPR) and we are struggling to insert plasmids in It. This specific transformation was made to insert a third plasmid in the yeast
I screened the potentially positive colonies by colony PCR and I saw no bands, whereas I got amplification of fragments of interest for the other previous plasmids. What would be the problem?
The reaction? Maybe a contaminant containing the same marker selection as my desired plasmid?
The primers for this reaction have the same Tm as the other ones that were successful. Do you guys have any suggestion? Thank you.
A few possibilities come to mind that you might wish to test for:
1) the colonies you got are actually contaminants of some sort (but less likely if they are giving you positive PCR products)
2) the colonies aren't actually real and if you restreak on selective media the phenotype will go away
2) that auxotrophic marker is able to revert at some low frequency and the colonies you got are revertants and not transformants
3) they are real and the problem is with your PCR. Did you do positive control with those primers and a different strain carrying that same plasmid?
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