Electroporation

Brian P Higgins via methods%40net.bio.net (by higginsb78 from gmail.com)
Wed Jan 28 10:37:11 EST 2009


You could try drop-dialyzing the sample on a 0.22 or 0.45 micron filter
first.  The filters come as small, 1-inch diameter circles.  I partially
fill a petri dish with ddh20, then drop the filter in.  I then put 3-5ul of
the ligation reaction (after deactivation) on the filter and let it sit for
5-10min.  After that, I pick up 1ul and transform via electroporation.  This
should work.  Some ligase mixes (like NEB's QuickLigase) contain Ligases
that can't be heat inactivated, so a column or precipitation would be
better.  In addition, some include PEG and other constituents that can't be
removed easily, but will hinder the transformation if not taken out.  You
can try a column spin as well, but the recovery is probably comparable to a
precipitation.  I sometimes use the Zymo clean and concentrator or the
Qiagen Minelute kits b/c they have very small elution volumes (<10ul).


On Wed, Jan 28, 2009 at 1:15 AM, yoginee budhkar <eenigoy from gmail.com> wrote:

> Dear All,
>
> I want to use biorad gene pulser for transforming *E. coli* with my ligated
> mix.
>
> It is mentioned in the bio rad manual that we must incubate the ligated mix
> at 65deg C for 15 min to inactivate the enzyme and then proceed with its
> dilution or etOH precipitation to get rid of extra salts that may decrease
> the resistance and cause arcing.
>
> I did go for the boiling step, but I'm afraid I'll lose the DNA by
> precipitation since already only about 80 to 100 ng, insert + vector DNA is
> present in my ligated mix. Cannot afford to lose any of it during
> precipitation
>
> What could be done to get rid of the salt content that hampers the
> electroporation?
>
> --
> --
> Yoginee Budhkar
> Junior Research Fellow
> Food Engineering and Technology Department
> Institute of Chemical Technology
> Matunga, Mumbai 400019
> Contact: +91 9223355677
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