Now I want to do a gene disruption in the yeast strain SEY6210. The mark gene was LEU2. Positive clones were identified by PCR (Primers were designed at both ends of the target gene). PCR results showed that the target gene was not disrupted correctly. But the clones can grow on the SD-LEU medium. Does this result mean that the LEU2 gene integrated into the LEU2 locus? If so, how can I avoid this situation?
you simply have to check more colonies and increase the homology length if you want more accurate integration. This does not necessarily mean that the marker integrated in its natural position it could be elsewhere. If you used a large amount of cells and only could get very few transformants it might be revertants since this is not a full ORF deletion.
Hi Hanna Alalam, thanks for your Kindness. the homologous region is long enough, I inserted the LEU2 gene into the target gene, then cloned the target gene + LEU2. The PCR products were transformed into yeast competent cells. Can you teach me why the clones can grow on the selection medium even the PCR results were not correct?
I will check more clones, Thank you again
Just to be clear you had your target gene on a plasmid then you inserted the leu2 into that? then created A PCR cassette from that plasmid and transformed into yeast? if so what plasmid was it ? is capable of replicating in yeast? did you gel purify the insert before transforming? because it you had your cassette from a self replicating plasmid then you could be just transforming the cells with the plasmid rather than integrating.
Yeah, I put the target gene on plasmid PUC19, and insert the LEU2 in the middle of the target gene. the cassette contain the target gene and LEU2 was PCR amplified from the plasmid. The PCR product was purified by ethanol precipitation methods, then was transformed into yeast cells. PUC19 can not self-replicating in yeast. I want to disrupt the target gene, so I must integrate the LEU2 marker into the target gene in the chromosome.
Ok in that case it is more likely just be a random integration event and in that case you just do as I said above and screen more colonies
did you do the cloning in a way to remove a part of the target gene. or was it just an insertion of LEU2 into a single restriction site within your target gene?
another remark: is it likely that the disruption causes lethality or slow growth? this would mean that you have a strong selection for cells that do not have the construct correctly integrated.
I would try to delete your target gene in diploids, select for Leu+, check them by PCR and sporulate them.
another approach: if strain background is not important, go to the BY series of strains.
yet another approach: use other marker genes that have no homology in S. cerevisiae, such as kanMX, S.pombe his5 or others.
so you see there are possible solutions to your problem.
Hi Mingdong.
I would first make sure that disruption should be done in haploid mating type.
The diploid strain may result in heterozygous conditions where PCR may show the positive result for disruption (and as you say, PCR results showed that the target gene was not disrupted correctly), but that will also grow on selection media.
Whatsoever maybe, I would then go for sporulating the cells and dissecting the tetrads to pick the homogenous haploid spores and then confirming these spores for positive and clean disrutions.
Hi, J. Stolz, Thanks for your advice. I just insert the LEU2 gene into a restriction site within my target gene. The strain background is important for my assay. Next step, I want to follow your advice to use the selection marker kanMX.
Thanks again!
Hi, Amit, thanks. The mating type of the strain I used was a and I have double- check its mating type via PCR. No problem within this part. Thanks again.
