Searching desperately for pJM17 (E1 adenovirus)

noone notyet
Fri Aug 23 08:10:42 EST 1996


In article <trevor-1608960618330001 at snews.zippo.com>,
trevor at leland.stanford.edu (Trevor Bezdek) wrote:

>   So, I am desperately in need of a very large quantity of pJM17 DNA.  For
> clarification, pJM17 is the large adenovirus DNA with only an E1
> deletion.  It gives better gene promotion than the more available pBHG11,
> which has both E1 and E3 deletions.  So, I have some good DNA but all
> attempts at transformation seem to lose chunks of the insert (it is 40kb
> in a plasmid vector).  So does anybody have any suggestions as to how to
> make the transformation work or even better a commercial source (speed is
> important enough to justify a high price).
> 
> Thanks,
> Trevor


I have done this without any problems on a number of occasions.  I would
recommend that you:
1.  Use TG2 E.coli - these are deficient in some of the recombination
machinery, so their ability to take chunks out of PJM17 is reduced.
2.  Do the transformations/mini-preps/bulk-preps as fast as possible. ie
plate out transformants at the end of the day, pick colonies in the
morning, grow up mini-preps during the day for 5-6hrs and then grow the
bulk preps that evening for doing the next day.  The metabolic stress on
the bugs of all that plasmid DNA isn't good for 'em...
3.  Use the Qiagen maxi prep kits.  This is quick and doesn't require
passing the DNA through needles at any point.  (I don't work for Qiagen by
the way).
I have used PJM17 prepared this way to generate RAd's by homologous
recombination.
Good luck.
Tony



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