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Trouble with yeast two hybrid positives

Frederik [iso-8859-1] Börnke ricky_boernke at gmx.net
Wed May 2 05:16:09 EST 2001



subhash schrieb:
> 
> Hi,
>     I have been doing two hybrid screening with a whole length protein.
> I had go some His+ colonies and then used them for LacZ sceening.
> Positives from this screen were then checked using PCR with primers on
> either sides of MCS of the 'prey' plasmid. But In all of them I am
> getting a 200 basepair fragment- none of the His+, LacZ positive strains
> 
> are showing an insert in this PCR screen. I would like to know if
> anybody has experienced this kind of problem in yeast two hybrid
> sceening. I am using L-40 strain of yeast. As bait plasmid I have
> pBTM116 and the library is in pACT2. I really hope somebody can help me
> out. Thanks.
> Subhash
> subhash at ccmb.ap.nic.in



Hi,

we have observed the same phenomenon when doing colony pcr on
th-positives. We have cloned the 200bp band and sequenced it.
It turned out to be some yeast gene (can't remember what it was).
However, we were also able to amplify the insert of our AD-vector.
If you don't have to many colonies to check, just get on with the
regular protocol like plasmid rescue in E. coli, confirming the
interaction, seqeuencing so on and so forth.

Ricky 

 
*******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de





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