Problems of electroporation.

Sergio sergioal at bbm1.ucm.es
Thu Sep 7 13:02:51 EST 2000



Chris LaRosa wrote:

> Helen Mok wrote:
> >
> > Does anyone have experience of successful
> > transformation of control plasmid into E. coli with
> > electroporator, but nothing resulted with ligated DNA
> > (after ethanol precipitation)??
>
> How do you know that have ligated DNA (assuming ligated into a plasmid)?
>
> If your ligated DNA is not working then it indicates that you lost the
> ligation products during precipition or that the ligation failed.
>
> You can add c0-precipitants to pull down the ligation products... say 10
> ug of trna.

I agree. I would say that the ligation is not working or you are losing DNA
during the precipitation step. Check the ligation mix using another
competent cells or try a carrier to help in the precipitation.
Recently i read a interesting (and short) paper about electroporation, which
remarks the importance of the carrier:

Heng Zhu and Ralph A. Dean. "A novel method for increasing the
transformation efficiency of Escherichia coli-application for bacterial
artificial chromosome library construction". Nucleic Acids Research, 1999,
Vol. 27, No. 3, 910-911.

Sergio






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