Plasmid size limit for transformation

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Thu Oct 16 12:55:51 EST 1997


At 17:02 10/16/97 GMT, Torben =AFsterlund wrote:
>I am about to make a construct that will be approx. 21 kb (including the
>2.9 kb of the vector, pBluescript).
>My question is: will it be possible to transform E. Coli with this
>(monster) plasmid  by electroporation?. Should I adopt some special
>precautions???=20
> Thanks for your tips!

Juan:

If the construct is intact, it will be hard, but you can get it in and
electroporation is likely to be your only viable choice.

Just for fun, I prepped XL-1 mrf' for electroporation and took 10 ng of
various purified plasmids that I had and tested the efficiency.  With
bluescript and pUC19 both were just short of 10^8 (actually high 10^7
cfu/ug), with the old BlueBacIII (10.5kb), the efficiency dropped off to
the low 10^4 cfu/ug.  Finally, when I tried an intact cosmid (40-45kb), it
dropped further down into the low 10^3 cfu/ug.  So in short, it's do-able,
but you may have to struggle with it.

Hope this helps,
David




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