Hanahan competent E.coli

Martin Kennedy mkennedy at chmeds.ac.nz
Fri Apr 21 16:14:31 EST 1995


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

Well, I use a Hanahan protocol for DH5a whch gives us about 
5x10^7 to 2x10^8 cols per ug routinely, which I find good 
enough for most purposes. I inoculate 50ml of room temperature 
(not cold) 2xYT medium, containing 10mM each of MgCl2 and MgSO4 
(don't know if this is v. impt) with 0.5ml of an O/N culture.  
Then incubate with vigourous shaking for 2 to 2.5 hours, no 
longer.  This seems about right, and always gives good cells. If 
you want super good efficiency, better than 10^8, then you may 
need to do O.D. and viable counts etc, but I've never bothered.

> Finally, do XL1 blue cells give worse or better transformation frequencies?

I've never compared them directly (in the same prep), but  I've 
never noticed much difference (though I tend to use DH5a much 
more often).

I think it is important to use  a reasonably freshly streaked 
plate of cells to inoculate the O/N. Hanahan states that thou 
shalt use colonies directly off the plate to inoculate the larger 
culture, and growing an O/N  reduces efficiency.  Again, you may 
like to try this for better efficeincy.

Good luck...

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
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