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yst 2 hybrid: weird results

Karen Roberts leehrat at mindspring.com
Tue Nov 28 18:26:03 EST 2000


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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