Inserting a large DNA fragment into a large plasmid

Martin Offterdinger a8803349.nospam at unet.univie.ac.at
Fri Sep 17 01:58:18 EST 1999


On Thu, 16 Sep 1999 11:56:41 -0500, Paul Jasper
<pjasper at orion.luc.edu> wrote:

>
>Don't worry too much about getting large DNA fragments into E. coli.
>We transform 25kb plasmids into E. coli (by electroporation).... and it
>works just fine.  The transformation efficiency will be lower than
>normal but you should have no problem getting colonies.
>
>We've tried up to 50kb plasmids with success.  The only other thing I
>would suggest is to be very careful about growing conditions... Do not
>let the bugs overgrow!  (I usually go 12 hours) ... When the bugs
>overgrow, weird things happen, such as deletions and aggregation.
>
>
Another option would be to use a strain that has been specfifically
designed for large plasmids , such as XL10 Gold from Stratagene (no
affiliation) I didn´t try it, but they claim to get higher
transformation efficiencies for large plasmids with this strain.
martin
Martin Offterdinger
Internal Med.I,Dept. Oncology
University of Vienna
Austria
E-Mail:a8803349.nospam at unet.univie.ac.at
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