Hi i am phd student and i have started working on yeast two hybrid GAL4 system. After transformation i am not getting sufficient colonies and i also want to know that can direct spreading of transformants on -LTH plates affect transformation efficiency. As i am not getting colonies on these plates. Can you give me some guidelines for using this system and any precautions that should be taken.
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blastokar
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Posted 14 January 2010 - 12:40 PM
reenu, on Dec 2 2009, 07:17 AM, said:
Hi i am phd student and i have started working on yeast two hybrid GAL4 system. After transformation i am not getting sufficient colonies and i also want to know that can direct spreading of transformants on -LTH plates affect transformation efficiency. As i am not getting colonies on these plates. Can you give me some guidelines for using this system and any precautions that should be taken.
Hi, I have a little experience with two hybrid but maybe I can help you. I think that plating on -LTH can affect your transformation efficiency because of it is too much "work" for cells, to synthesize the necessary for growing without the three markers. It is better to plate on -LT and thereafter make replicates to -LTH or -LTHA (if A is a 4th marker). If you plate directly on -LTHA (non on -LTH) you can get less colonies but also less false positives. Check before testing your protein interactions autoactivation of preys and growing without H.
Good luck
