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
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