Hanahan competent E.coli
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
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.
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
NN NNNN Christchurch, New Zealand ZZZZZZZ
Phone (64-3)364-0880 Fax (64-3)364-0750
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