Hanahan competent E.coli

anagno at mpimg-berlin-dahlem.mpg.de anagno at mpimg-berlin-dahlem.mpg.de
Thu Apr 20 05:33:08 EST 1995


In article <3n2mqg$3i4 at mserv1.dl.ac.uk>, LOGAND <logand at msdos.montpellier.inra.
fr> writes:

> Hi,
> 
> I am planning to prepare competent cells by the Hanahan method to use for 
> the transformation of many ligation reactions (nested deletions prepared 
> using the Pharmacia kit). I have never used this transformation protocol 
> before, Sambrook et al. states that the number of cells in the culture 
> should not exceed 1x10^8/ml and one should determine the relationship 
> between OD and viable cell number/ml for the strain to be used. Well, I 
> would love to be able to omit (or at least limit) this time consumming step 
> - thus, can anyone give me a value for this using DH5a? I realise I will 
> have to check/re-calculate this due to differences in spectrophotometers 
> (light scattering etc) but at least I would have an approx. starting point.
> 
> Finally, do XL1 blue cells give worse or better transformation frequencies?
> 
> 
> Thanks
> 

Hi,
I have many times prepared successlully DH5a competents, cells. The best OD600
to collect them is at 0.3-0.4. It is better to be nearer 0.3 than 0.4 but
any OD between these two is OK. For the XL-1, I think that they sometimes
give a little better results in transformation, but not always. I suppose
depends on the weather.
You do not have to use the method in Maniatis. A simpler method gives the
same results. It is the one described in Promega's manual for the Altered
Sites II mutagenesis kit, and it works equally well. If you do not have it
and cannot find it, you can send me a private mail, and I will send it
to you.
The easier protocol to use is the simple CaCl2 method described also in
Maniatis. It gives slightly worse results than either the TFB or the 
Promega method, but for normal cloning purposes it is more than enough,
unless you have something hard to transform.
The BEST method for the transformation of E. coli is electroporation.
I have recently tried it with DH5a and it gives much better results that
any of the other methods. I plan to abandon the other methods for the sake
of electroporation. It is also much easier to perform. If you can have 
access to an electroporaton apparatus and you need maximal transformation
efficiency, this is the best method.

Bye

Kostas Anagnostopoulos
<anagno at mpimg-berlin-dahlem.mpg.de>
 



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