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Ultracompetent E.coli cells!?! - Summary

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Wed Dec 13 04:12:34 EST 1995


In article <4al7t2$3bb at epx.cis.umn.edu>, shin at biosci.cbs.umn.edu (Shin
Enomoto) wrote:

> : I«ve asked one person from Stratagene, and he told me, that they are using
> : a modified Inoue-protocol. But he dindn«t want to tell WHAT they are doing
> : with the cells.
> 
> I personally don't need anymore convincing arguments to convert to 
> Inoue-protocol.

Ditto

>  Rather than waiting for another five years for somebody to optimize and
> push the limit of competency, can we keep posting on the net minior
> modifications or oops that gave competent cells 10^10. Even if individual
> attempts do not amount to statistically significant results, maybe as the
> bionet collective we can find a significant improvement that is not
> private.

OK.  One of the things that Inoue et al do not address is the inclusion of
reducing agents in the transformation reactions.  Stratagene recommends
adding beta-mercaptoethanol to 25mM to the reactions.  I once did a
comparison with BL21(DE3)plysS, prepared with the Inoue method (grown at
23*C though), transforming with a pET derivative.  I found that the
mercaptoethanol increased the transformation frequency five fold and
adding DTT to 17mM resulted in 20 x more colonies.  No small increase. 
Since then I always add DTT to my tranformations if I need high
efficiencies.
  Of course this is the result of a single experiment so the statistics
are not exact and this may even have been a coincidence, but anyway, there
must be some reason for Stratagene to recommend b-me.  If someone is
willing to look into this I would be very intrested to hear of the
results.

Zophonias

_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_____________________________________________________________________



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