What E.coli strain should I use?

Ashok Mudgapalli ashok at LAMAR.COLOSTATE.EDU
Thu Oct 19 21:30:10 EST 1995


Dear Netters
	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

Sincerely

Dr.Ashok Mudgapalli



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