low LPS plasmid prep protocol

Nick Theodorakis via methods%40net.bio.net (by nick.theodorakis from gmail.com)
Mon Aug 24 21:13:14 EST 2009


On Aug 24, 8:50 pm, Aawara Chowdhury <aaw... from pontiff-playground.org>
wrote:

[...]
>
> I didn't know that all nucleofactor had was a peptide - that's
> worth trying.  Many years ago I was told that some of the nucleofactor
> buffers contain a low level of wortmannin. ...

Wow, seriously? And they didn't disclose that? You'd think people
would want to know something like that.

High copy plasmids are great when you can use them. I used to max out
the Qiagen Maxi preps (> 500 ug) from 50 ml superbroth culture using
Bluescript-derived vectors, and even more yield from a CsCl prep back
when I did them (we could often pull the band just viewing under room
lighting).

> The idea being that transfected DNA contains nicks etc. that activate ATM - possibly
> resulting in apoptosis of transfected cells.

It seems in that case then one advantage of using CsCl is that one can
grab only the supercoiled form and forget the nicked version.

I thought I remembered reading that nicked forms are more prevalent
when using chloramphenicol amplification (as one might when using
pBR).

Nick

--
Nick Theodorakis
nick_theodorakis from hotmail.com
contact form:
http://theodorakis.net/contact.html



More information about the Methods mailing list