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-   -   Problem with Isolating Library Plasmid from Yeast (http://www.molecularstation.com/forum/dna-techniques/77558-problem-isolating-library-plasmid-yeast.html)

khan_bgu 04-04-2011 08:34 PM

Problem with Isolating Library Plasmid from Yeast
 
am a PhD student from Israel.In our lab we did Yeast Two Hybrid using the Matchmaker Gold Yeast Two Hybrid Kit from Clontech.To minimize the frequency of false positives we also used Normalized Arabidopsis Library from Clontech.
Hello All.
I In short my problem is that I am not able to isolate the interacting protein containing the Library plasmid from yeast.I did mating between the Y2H Gold strain containing my bait with library in Y187 strain with Prey.I got nice single blue colonies on SD-leu-trp/X--alpha-Gal Media and also on Quadruple Drop Out Media as well.
But as of now I have not been able to either Isolate Plasmid from these clones or do colony PCR.I get primer dimer always when I do colony PCR whereas when I make plasmids using Zymo Mini Prep kit and transform into E.coli I get a very high band more than 15KB suggesting its Yeast genomic DNA.I have tried also plating those clones on SD-LEU media for selecting library plasmid and then using very less amount (about 5 mg pellet) to make plasmid as suggested by Clontech and doing electroporation but still got Genomic DNA on Gel.
I am really stuck and dont know what went wrong as I did everything according to protocol.
If anyone had this kind os difficulty working with Yeast and would share it would be great help.
I dont know if I should do the assay again as I am afraid that I would get the same results again.
Any help highly appreciated
Thanks
Asif

anubis2005 05-28-2011 03:37 AM

Re: Problem with Isolating Library Plasmid from Yeast
 
Hi,

I saw your post about not being able to isolate the plasmid from your yeast. I'm also pretty bit perplexed about why you weren't able isolate the prey vector. I was wondering if you managed to figure it out, as I'll be performing a Y-2-H soon.

khan_bgu 05-30-2011 01:56 PM

Re: Problem with Isolating Library Plasmid from Yeast
 
Dear Anu
Hi !!
I did manage do isolate library plasmid after all.
I used the protocol from clontech Protocols Handbook for plasmid isolation from Yeast.
You can find it on the internet just type Clontech Yeast Protocols Handbook.
Good Luck
Asif

hairongxu88 06-18-2012 06:42 AM

Re: Problem with Isolating Library Plasmid from Yeast
 
thanks very much


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