YACs

Andrei Popov andrei.popov at bbsrc.ac.uk
Sat Nov 16 16:46:14 EST 1996


David Hills wrote:
> 
> Trying to make a YAC library (pamphlet so far!) and getting very few
> successful transformations. Does anyone have any hints and tips? Does
> anybody recommend any particular supplier for Sorbitol and PEG 6000??
> Thanks,
> Dave Hills
> Roslin Inst.
> Edinburgh

It was not easy for anyone.
Sorbitol- Fluka 85530 (yes, the most expensive)
PEG 8000- Sigma P-5413
Tubes for transformation- Falcon 2059
Water- BDH mol. biol. grade
VACA without amino acids (Difco) can also help.

There are two main tricks: ensure that the ligated DNA is
not degraded (PFGE) and play around with the amount of Zymolyase
(or whatever enzyme you use) per defined amount of cells.
The enzyme must be aliquoted and stored frozen. 

The amount of enzyme must be titrated for yeast collected
EVERY time at the SAME STAGE of growth. What else...
polyamines were reported to improve the chances to recover
bigger YACs.

Best wishes, and do not despair!

Andrei



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