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Yeast Two Hybrid screen MAPK3K


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#1 Signal

Signal

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Posted 27 December 2009 - 07:24 PM

Hello,

I'm performing yeast two hybrid for a protein. My problem is that no one from my lab has done this before and the Prof is too busy to guide me in detail.

Using clonetech system I've prepared all the cDNA libraries and have done transformation of bait into strain AH-109 using GEITZ lab protocol and its doing well so far.

I'm going to write the method here along with some questions, please rectify me;

1) Do I need to make a stock of bait+AH109 and use it further for co-transformation with the prey?

2) After co-transformation with the prey using SD-DDO media, I'm supposed to streak the colonies from SD-DDO on SD-TDO and QDO medias but how to use 3-AT to control back ground growth and what is this background growth, are these non-transformants/false possitives?

3) Is it enough if I screen only on SD-TDO and SD-QDO medias to report the weak and strong interactions or I should also use X-aGal?

4) Is there any calculation involved in the analysis of data?

Pardon me for getting you in too much detail :rolleyes: .

Cheers
I/we have to wait for 30 years to let the mirAcle haPPen....someone here told me!!!!




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