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Large insert cloning

Sendil Devadas skd4 at psu.edu
Tue Mar 7 18:04:19 EST 2000


Dear folks,
I am having trouble cloning a 100 kb BAC insert into a TAC vector (22
kb) for eventual complementation. Any  suggestion regarding DNA
isolation ..digestion..ligation and transformation will be very helpful
for me. I supplement my digestion with spermidine, BSA, DTT and do a
B-agarase treatment of my low melting CHEF gel and drop dialyse my
ligation mix. My vector is CIP treated and I use 10:1 (vector:insert)
for ligation, supplemented with ATP. I follow the Gibco-BRl
electroporator protocol for transforming DH10b cells.
Advance thanks for your help
Regards

Sendil Devadas

Graduate Student
Program in Plant Physiology
Penn State University
PA 16802


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