Pichia pastoris _problem zeocine resistence - (Sep/01/2010 )
Hi
I try to express some protein in Pichia pastoris.
And I have the problem: when I carry out electroporation and spread yeast on YPD plates with zeocine ( 100ug/ml, 500ug/ml ), yeast grow only one day and the plate is full of yeast no colonies just coating. I tried to spread P.pastoris without vector contained zeocine resistence ( pPICZalpha)like negativ control. And the plate is coating again. The same I have when I spread E.coli nova blue or TOP10 on the LB low salt medium plates containing zeocine. I changed zeocine,
I controled preparing of medium and the result is same.
I do not know what I do now.
Could you help me somebody?
Thank you very much
-sius-
sius on Wed Sep 1 12:02:21 2010 said:
Hi
I try to express some protein in Pichia pastoris.
And I have the problem: when I carry out electroporation and spread yeast on YPD plates with zeocine ( 100ug/ml, 500ug/ml ), yeast grow only one day and the plate is full of yeast no colonies just coating. I tried to spread P.pastoris without vector contained zeocine resistence ( pPICZalpha)like negativ control. And the plate is coating again. The same I have when I spread E.coli nova blue or TOP10 on the LB low salt medium plates containing zeocine. I changed zeocine,
I controled preparing of medium and the result is same.
I do not know what I do now.
Could you help me somebody?
Thank you very much
HI,
I do not know which temperature you usually add the zeocin but is better if this temperature is less than 55 ºC.Are the Zeocin plate fresh plates? I mean it is better if the plates have less than a week. And the zeocin is ligth sensitive.
I hope this can help you
-SUVEAR-