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dysbindin

Member Since 09 Jul 2012
Offline Last Active Aug 11 2012 01:27 AM

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neurobiology

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malgosia18
10 Jul 2012 - 08:40

Topics I've Started

About gateway cDNA library

26 July 2012 - 01:02 PM

Hi, everyone,

I am looking forward to a methods, which can generate a good cDNA library into plenti6/V5 vector.

Right now, i have a cDNA library in pCMV-SPORT6, which have a attB sequence. I would like to use BP reaction to transfer the cDNA library to pDOnor vector, then transfer again by LR reaction to plenti6/V5 vector.

I would like to know is there anyone once play with cDNA library before? especially in the gateway system, as I would like to findout is there any publications using the two steps stratyge to generate a very nice library?

Best

dysbindin

Help on the yeast two hybrid

09 July 2012 - 06:03 PM

Hi everyone,

I am trying to use yeast two hybrid system to screen some interaction proteins. But I have big problem on the bait expreesion. I cloned the full length cDNA with start coden into pGBKT7 vector. The transformed yeast with this plasmid could growth on the -Leu SD plat, which means the vector were transformed into yeast strain. However, I can not detect the any expression bait by western, wheras, the pGBKT7-p53 could be detect. I am sure that my cloning work is right , as the sequencing shows no frame shit happened. Does anyone met this problem before? Can I use this bait to continue my screening?