What E.coli strain should I use?
hbenes at BIOMED.UAMS.EDU
Fri Oct 27 20:28:20 EST 1995
Dear Dr. Mudgapalli,
We have used the CaSpeR vector very successfully for several years in my
laboratory. We have had no bacterial transformation problem with an old E.
coli strain, HB101. It may be that your vector DNA comes from a different
bacterial strain than DH5 alpha or XL1-Blue that you are trying to use. So
find out first where your particular CaSpeR DNA comes from.
In the future, please give an e-mail address to answer you. My message,
which is intended to help you, will now end up with the BIOSCI
administrators who don't need to be distributing messages.
Good luck with your transformations, bacterial and fly.
Dr. Helen Benes
Department of Biochemistry/Molecular Biology
University of Arkansas for Medical Sciences, Slot 516
4301 West Markham St.
Little Rock, AR 72205
Tel.: (501) 686-5782
FAX: (501) 686-8169
E-mail: hbenes at biomed.uams.edu
Subject: What E.coli strain should I use?
Date: Thursday, October 19, 1995 7:30PM
I am trying to subclone some DNA fragments (12-20 Kb) in
Drosophila germ line transformation vector, CaSpeR. The size of this
vector is 7855 bp with white gene in it. I am having hard time in getting
the ligated plasmid intact after transforming the bacteria. I used ligation
kits from Boeringer M, 5prime-3prime, NewEngland Biolabs. All the kits
are working well because I could subclone about 7 DNA fragments (3-9 Kb)
in blue script without any problem. I used either DH5 alpha or XL1-Blue
cells for transformation. These both strains are working with blue script
but not with CaSpeR.
Since the CaSpeR has invert repeat sequences, I suspect there is
lot of rearrangement and restriction going on in the bacteria. Though
these bacteria are rec-, I do not understand why I am getting the DNA
fragments in between the sizes of vector and insert (after restriction
digestion). I am losing major portion of the ligated construct after
transformation. I am preparing the plasmid from transformants using
mini-boiling prep method.
I know lot of labs are using CaSpeR as a transformation vector
with their DNA fragment in it. I would greatly appreciate if some body
could give me the details of their ligation protocol and the strains of
the E.Coli used for successful transformation.
Many thanks to every body in advance
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