Making TOP10 e.coli competent

Emir Khatipov khatipovNO at NOuchicago.edu
Wed Apr 17 14:17:17 EST 2002


I don't use DMSO (Inoue method), but RbCl method (see e.g.
http://research.nwfsc.noaa.gov/protocols/rbcl.html ) worked always fine for
me with all E.coli cells I used so far, including Top10. We discussed this
issue in this group many times in the past. I am not sure why DMSO method
does not work for all strains, but it probably has something to do with
oxidative nature of DMSO itself. Some strains might not survive oxidative
stress unless lower concentrations of DMSO are used ---- just a thought.
Emir

"remove the [nospam] (Nicolas von Ahsen)" <nvahsen at hot[nospam]mail.com>
wrote in message news:3cbdb293.2384482 at news.gwdg.de...
> Dear Netters,
>
> has anyone been successful to make TOP10 or TOP10F (from Invitrogen)
> cells competent again? I tried it myself (Inoue method) and got cells
> of very low competency. I also heard a rumour that these cells cannot
> be made competent again for some strange reason (but I cannot
> generally believe this, e.coli has no memory).
> And why do I want TOP10? Because I tried preps from identical plasmids
> in parallel from DH5a, XL1blue, TOP10 and TOP10F and I always got more
> plasmid from the TOP10 cells. Competency of my DH5a & XL1blue is fine.
>
>
> Any hints?
>
> Thank you
> Nicolas
> -----------------------------------------
> University Hospital Goettingen
> Internal Medicine
> Dept. Klinische Chemie/Clinical Chemistry
> Dr. med. Nicolas von Ahsen
> Robert-Koch-Str. 40
> 37075 Goettingen
> Germany
>





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