Is there any chance that your "retesting" system makes the protein of the clone
lethally toxic that killed the host yeast cells? The expression level
difference (higher) may make an otherwise less toxic protein lethal to yeast.
Zhonglin Chai
Karen Roberts wrote:
> Hi all,
>> I am sceening a mouse brain library for interaction with our bait. For
> this particular clone (call it B60),I made the lysate, did pcr with AD
> plasmid primers to reveal an insert. I then sequenced the pcr product.
>> Next I made a miniprep of the clone and sequenced to confirm the same id
> as for the pcr product; same identity came up.
>> NOw my problem, in retesting interaction b/t this clone and our related
> bait as well as unrelated baits, I can't get -LT txfmts and only a few
> -leu txfor.
>> I cleaned up the miniprep; no differnce. Next, I started from scratch
> and made a new lysate, did pcr, new miniprep, etc.
>> I still don't get transformants. I am doing the procedure right b/c my
> controls worked beautifully. So it's something about this dna;any
> idea??
>> Also, I sequenced this second B60 miniprep to test version 2 of big dye
> terminator kit. Thesequence data was beautiful but the blast search gave
> me a complete out of left field identity. How can this be when it is
> the same clone as the origina B60?
>> I feel like I have run out of ideas to make this work. oh, I also
> repeated the transformations with double the dna as the first time;
> still next to no or no txfts.
>> Can anyone help me? I've only learnd about the yeast 2 hybrid system 6
> months ago so I am not by any means an expert
>> Thanks
> Karen
>
--
ZhongLin Chai, PhD
__________________________________________________________
Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital
Commercial Rd, Prahran, VIC 3181, AUSTRALIA
Telephone: (61 3) 9903 0698 (lab)
(61 3) 9903 0696 (office)
Mobile: 0413 58 1940 or International: +61 413 58 1940
Fax: (61 3) 9903 0731
email: zhonglin.chai at med.monash.edu.au
___________________________________________________________
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