I have been trying to integrate a DNA cassette into the CAN1 locus using canavinine counterselection, however, I am getting problems with getting any colonies.
Details:
Strain: BY4741 .
Counterselection media: -ARG + 60ug/ml canavinine.
transformation method: standard lithium acetate transformation.
I am using a standard canavinine counterselection concentration mentioned in the literature for this strain, however, the transformation is failing to yield any colonies on both the negative control and the transformation plate (I expect with this kind of selection you will have colonies on the negative control due mutation in CAN1 locus not related to the DNA cassette, but you will simply have a higher number of colonies on the transformation plate). I have checked that the plates are able to support growth of the yeast by streaking a CAN1+ and CAN1- strains on my plates and the results were as expected (the CAN- strain grew and the CAN1+ strain did not). I also attempted the transformation again but included a 2 hour recovery step in YPD prior to plating instead of direct plate and that still did not work.
Any ideas on how to troubleshoot this further?
/Hanna
Dear Hanna,
I may have some suggestions.
1. Try to make a plate with lower concentration of canavinine, seed the cells on them followed by replica plating to the plates with 60ug/ml , to identify the true colonies.
2. Try to make overnight culture of the transformed cells, dilute and seed on several plates with 60ug/ml. In that case, if there still Can1p is expressed is will be degraded in cells with real integration.
Hope that is helpfull and makes sense to you
Best Regards,
Dima
Dmitry Zabezhinsky Thank you very much for the suggestion I will give it a go and see how it goes.
/Hanna
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