I have been using Takara's Yeast Transformation System 2. Using the kit's protocol, I transformed Saccharomyces cerevisiae with plasmids that have auxotrophic selection (trp and leu) in them. I did spread 100 ul of 1/10 and 1/100 dilutions of transformed cells on respective SD/-trp and SD/-leu plates. After 5 days of incubation at 30C, nothing appeared on the plates.
I was wondering, if after transformation and recovery in the YPD Plus medium (provided with the kit), if I wash the cells with 0.9% NaCl and grow for 5-8 hours in the SD/-trp and SD/-leu broth, before plating on the SD/-trp and SD/-leu agar, will it make a difference?
Kindly also let me know, what all of you look to change when your transformations fail.
Regards
Hi there,
Selection by auxotrophy marker doesn't require any "recovery step" prior plating onto selective media. Recovery on rich medium is only required when using antibiotics resistance markers.
Dominique Liger is correct that you don't need an outgrowth step. The problem is therefore either in the technique or the plasmid. You might wish to repeat the experiment again and also try a known control plasmid DNA that you are certain is actually good DNA.
Hi again,
To go further into the analysis: there is either something wrong with the plates (recipe or mix up between the 2 sorts of plates) or an issue with the plasmids or with the transformation protocol. Did you grow cells on SD minus aa supplemented with aa? This should tell about the plate... For the plasmid issue, Michael already stated above... And about the protocol, make sure reagents are freshly prepared...
I have nothing to add to the comments above, which are right. You really need a positive control to show that both the transformation and the media are correct.
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