I've performed URA3 deletion in S. cerevisiae S288C by KANMX6 replacement. Transformants are able to grow on YPD containing 150 ug/ml G418 but not for the control (S288c). These transformats should not grow on SD due to URA3 was deleted. Unfortunately, these transformant are still grow on SD plate. After check with PCR using 500bp up/down primers, there are 2 PCR products occured which are 1.8 kb (original URA3) and 2.5 kb (KANMX). How can i overcome this problem and completely remove URA3 from its genome due to I have to trasfrom other plasmid with URA3 selection marker. Thank you.
Hi Natthaporn,
Well ... to test an auxotrophy is not carried out by streaking on synthetic medium but on a synthetic medium lacking this molecule or that molecule. So, you should assay your strain by spreading it onto a synthtetic medium not containing uracil, a so-called ura- SD medium. In other words, the fact that your strain grows on a synthetic medium does not necessiraly means that it is [Ura+] !
Best regards,
Pr Philippe Urban
In addition, if you get both the wildtype band and the "deleted" band in your test PCR, this indicates that you either have a mixture of clone’s or that your clones are diploid! So first tell if your strain is haploid (and has the HO nuclease disrupted)? Alternatively, is it diploid from the start? If the HO nuclease is not somehow inactivated, the strain spontaneously changes mating type and can virtually mate with itself, and generate diploids.
Curt
Hi there,
S288C strain being haploid, you are certainly confronted with a clone made of a mixture of wild type and mutated cells. I think the best is to streak the clone on geneticin containing media in order to isolate new and pure clones to be tested by PCR and by plating on SD-ura.
Hi again,
s288c is the strain used for the first yeast genome sequencing. For info:
Genotype: MATα SUC2 gal2 mal2 mel flo1 flo8-1 hap1 ho bio1 bio6
http://www.yeastgenome.org/strain/S288C/overview
Hi Dominique
So it is a stable MATalpha strain (ho is inactivated). So restreaking seems the best (and easyest) way to go, and test again. Streak on YPD G418 plates as you suggest.
Curt
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