[Methods-and-reagents] promoter mutagenesis question
novalidaddress at nurfuerspam.de
Sun Jun 5 16:29:51 EST 2005
Dear fellow Experts,
please give me advice on solving this problem:
I am about to assemble a mammalian expression vector. It contains the EF-1
(elongation factor 1) promoter. Unfortunateley, the promoter has a Xho I
site I need to remove.
I am thinking of two strategies:
1) cut with XhoI, do Klenow fill-in and re-ligate (in past experience,
there will be a lot of XhoI cleavable background)
2) perform site directed mutagenesis with a primer containing 6 wobble
bases (giving 4096 possibilities of new sequence). In this case, I'd
transfect cells with EGFP as reporter gene and screen them for expression
levels by fluometry of FACS.
Which is better in your opinion or what else would you suggest?
Wolfgang Schechinger, Dr. rer. nat.
Med Clinic I, Endocrine Research Lab
Bochum University Hospital "Bergmannsheil"
- the world's first casualty hospital for miners and steel workes -
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