"Re: wrong orientation of ligated fragment"

Narayanan Karthikeyan karthi at CCVAX.SINICA.EDU.TW
Mon Jun 14 11:16:26 EST 1999


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Dear all:
Me too have the same probelm, mine is little bit different than the
posted one here. I need to clone my gene at the EcoRI site of the Tet-on
vector and pSG5 vector. I need both sense and anti snese for
overexpression. All I get is the sense expression. I wonder whether the
anti sense is lethal to E.coli (I use Novablue cells). The stange thing
is, the novablue cells are grown on AMP+TET plates, i wonder this would
triger the expression in a TET-ON system. The other thing is the pSG5
expression vector has SV40 and T7 promoters. So now I am going to use
another enzyme (EcoRI and SacII) to cut out the insert from pSG5 and
clone into SacII and EcoRI site of the TET-ON plasmid. I hope this will
work out. The basic information of my message is sometime vectors has
both mammalian and prokaryotic promoters in it. So check that before
going into the cloning.
I hope the information is useful
sincerely
 
*************************************************** 
Narayanan Karthikeyan , Ph.D.,
Instructor
Department of Biochemistry and Molecular Biology,
East Tennessee State University,
Tennessee 37614
Tel: 423 439-8009
Fax: 423 439-8010
Email: narayana at etsu.edu
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