rapid ligation &electroporation

Frank O. Fackelmayer Frank at Fackelmayer.de
Fri Mar 23 13:15:16 EST 2001



Thomas Nilsson wrote:
> 
> Hello,
> 
> I am about to do a ligation using the Roche (previously Boehringer) rapid
> ligation kit , followed by electroporation of the ligation product. Roches
> manual states that the ligation reaction has to be purified with the "High
> Pure PCR Product purifiaction kit". Why is this? A component in the
> ligation kit interfering with the electroporation? Anybody with experience
> of electroporating rapid ligations?
> 

most probably to get rid of the salts and other ingredients of the
ligation mix, thereby avoiding arching during electroporation. This is
not different for conventional ligations, but EtOH-precipitation of the
ligation mix is usually sufficient. But add water to 100ul before
precipitating with NaOAC and EtOH, the DNA will be much cleaner and give
more clones than DNA recovered by precipitating in the small volume of
the ligation.

hope this helps,
Frank




More information about the Methods mailing list