Hi, I am working with a yeast strain which is knockout for a gene of interest (AMP Deaminase) (gene replaced by g418 casette). this strain cannot grow on S-adenosyl methionine (SAM). I transformed this yeast strain with a PCR product coding for the same gene from different organism so that the G418 casette is replaced by homologus recombination. I selected for the transformants on minimal media plate containing SAM. i got colonies after two days. Although these colonies are growing on SAM plate, when i streak on G418 plate, they grow there as well. And PCR indicates the presence of G418 casette even after transformation. If G418 casette has not been replaced why do i see growth on SAM plates. Are there some mutants that are unable to take up SAM? what might be happening? the strain and the reagents have been checked multiple times and the controls are working fine. Thanks in advance
Hi there,
Reversion is very unlikely as you perform gene knockout by full deletion. Therefore I think insertion of the PCR product might happen elsewhere in the genome explaining the persistence of resistance as well as the "reversion" of SAM use deficiency. PCR screens on transformants for location of G418 cassette and the presence of foreign gene would actually tell you.
I have done pcr for the gene of interest and also western...both were negative....
- Badges
- Science method