What E.coli strain should I use?
ashok at LAMAR.COLOSTATE.EDU
Thu Oct 19 21:30:10 EST 1995
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
More information about the Dros