Colony lift filter assay - (Jan/28/2009 )

Hi Everyone,

I am using Y2H analysis to check the interaction between two known proteins(alread shown to be interacting). I have the co transformed bait and prey plasmids in the GAL4 based yeast system which have lac z reporter alone. So I have done the colony lift filter b-galactosidase assay and found 5 out of 50 cotransformants to be +ve. But it is too low to be detected for such a known interaction. I was immersing the filter containing the colonies for about 20 seconds in liquid nitrogen and I am following the clonetech protocol.

Now my question is: Whether the cells were not crack opened in that 20 sec? Can I immerse in LN for a longer duration if so how long?

I am submitting this in this forum so that the experienced may help finding a solution.

Thanks in advance.

Well sorry but I don't really understand your question can you give more details