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yeast two hybrid - strange results


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#1 n2702582

n2702582

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Posted 22 September 2004 - 06:11 AM

Sorry if this is a bit long winded!
I have carried out a yeast two-hybrid screen using the Matchmaker system (Clontech). The MATa yeast strain, Saccharomyces cerevisiae AH109 was transformed with pBD-InvSpag (my construct - shorter version of inversin protein) by LiAC transformation. The yeast two-hybrid screen was performed using a pretransformed mouse 17-day embryo Matchmaker cDNA library(pACT2AD) (Clontech) and interacting clones identified by the ability to grow on minimal SD agar medium lacking tryptophan, leucine, histidine and adenine. After re-streaking on QDO I had 271 colonies, I plasmid rescued these and miniprepped and sequenced them. From these I identified 30 named proteins. I then carried out tests to eliminate false positives. they did not interact with unrelated bait, empty vector or autoactivate the reporter gene. However when tested with original bait (pBDInvSpag) the interaction had disappeared. To confuse me even more when tested against the full length protein six of them interacted even though they did not interact with the construct that was used to pull them out. Does anyone have any suggestions? Do the results that interact with the full length protein mean anything? HELP!!!




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