M13 sequencing templates from XL1Blue

Rosemary T. Hoffman rth at anat.UMSMED.EDU
Mon Mar 22 08:44:54 EST 1993


I am trying to sequence a series of Exonuclease III deletion constructs
which I have made in M13BM20 (Boehringer Manheim's new version of mp18).

I am using XL1-Blue as a host rather than JM109 because of its ease of
selection with tetracycline, however I'm having trouble getting clean
sequences.  XL1-Blue seems to grow faster than JM109 and my advisor was
concerned that I had too much chromosomal DNA in my overnight supernates.

I have tried 8 hour cultures, while purer on agarose gels the pattern seen
on sequencing gels is still dirty.

I do occasionally get a good clean sequence, but this is the exception
rather than the rule.


Has anyone had success with growing M13 in XL1-Blue for this purpose?
BTW, my largest insert is 2.5kb so I don't think I'm losing insert -
however I am going to check for this.
Thanks.

Rosemary



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