40kb Electroporation

Dima Klenchin klenchin at macc.wisc.edu
Sat Sep 9 20:44:24 EST 1995


In article <BARRYM.95Sep8144806 at stella.med.utah.edu>,
   barrym at stella.med.utah.edu (Barry Moore) wrote:
>
>
>I am trying to move ~40kb inserts from Scos to another vector.
>Can anyone give me advice or point me to references on transforming
>large kb (~50 kb with vector) DNA.  I am currently using E. coli DH10B,
>BioRad Gene Pulser, 1.7 kV, 25 uF, 100 Ohms, 0.1 cm cuvettes.

1. Leonardo, Sedivy: BioTechnology, 1990, 8(9):841-4  (5-150 kb, E.coli)
2. Mozo, Hooykaas: J.Plant Mol. Biol., 1991, 16(6):917-8 (~200 kb,
	not sure about bacterium)

I think (not sure) there also was a note in NAR about ~200 kb in E.coli

Don't see anything wrong with parameters. Apart from non-electroporation 
related problems the only possibility: DNA denatures in too high
el. field strenght (can lookup ref., but it's in Russian), forming "clots".
(DNA conc. should be high enough, though). Try more concervative
settings - 2.5 kV/0.2 cm, 25 uM, 200 Ohms. The number of transformants 
is usually lower, but worth trying. 

Hope it helps. - Dima



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