M13 sequencing templates from XL1Blue

Bruce Roe broe at aardvark.ucs.uoknor.edu
Tue Mar 23 08:09:00 EST 1993


In article <1993Mar22.134454.13862 at anat.UMSMED.EDU>, rth at anat.UMSMED.EDU (Rosemary T. Hoffman) writes...
> 
>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
> 
Rosemary,
	We are using XL1-Blue-METH, mainly because it lacks EcoK, and
it yields very clean ss M13 templates.  Thus, a couple of things for
you to look at.
1. Can you isolate and get good sequence from M13BM20 containing XL1-Blue
   alone?  This will test your isolation procedure, growth conditions,
   sequencing reactions, etc.  DO THIS CONTROL FIRST!! and GET THIS TO
   WORK before fooling with anything else.
2. Are you doing a second spin to initially remove cells before the PEG
   pptn step to concentrate phage?  Some leave the cells in the cold room
   overnight and forget to spin down again, which causes chromosomal
   DNA contamination in the final M13 prep.
3. The presence of Tet maintains the F-factor and is easier to deal with
   than having to always pick fresh from minimal media plates.  Thus,
   the original growth of XL1-Blue is on Tet plates and we then grow
   from there on out in the liquid media recommended by Stratagene (sorry
   I'm home and don't have the details handy).  Contact me if you can't
   find the details.
Cheers.....bruce
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