Problems with pBabe - unable to cut out the insert...

Ilya Shamovsky shamovsk at techunix.technion.ac.il
Fri Aug 18 03:21:26 EST 2000


Dear Netters,

The problem is this: we received pBabe  (puromycin resistance) plasmid
with an insert to be used in transfection of animal cells. The plasmid
is ok as evdent from restriction analysis and works fine in transfection
experiments. However, when we made this plasmid in E.coli, it did not
work in transfection. What is more strange is that it is no more cleaved
by appropriate restrictases. We tried a few strains (XL1, XL2, DH5
alpha) to no avail. At the same time, the same vector with different
insert seems to work just fine. How come bacteria can not tolerate this
particular DNA? Or is this a problem with plasmid isolation? (unlikely
since we tried a few mini- and midi-prep kits and again, it works fine
with another insert).

Any ideas?
Thanks in advance.

Ilya Shamovsky






More information about the Methods mailing list