Screning E.coli transformants ...

[Bernard P. Murray, PhD]

In article <79pv3v$kni$1 at nnrp1.dejanews.com>, Nick Theodorakis
<theodorn at gusun.georgetown.edu> wrote:

> In article <bpmurray*STUFFER*-0802991245480001 at mac-daddy.ucsf.edu>,
>   bpmurray*STUFFER*@socrates.ucsf.edu (Bernard P. Murray, PhD) wrote:
> 
> <snip>...
> > this method to work?  I have a couple of really nasty
> > ligations to perform at the moment and I am very tempted
> > to try this as I know the desired clone is there somewhere
> > (PCR) but many screeninge later I haven't found it
> > (one more round and it will have to be colony hybridisation).
> >      Thanks for any input,
> >           Bernard

> You have a higher tolerance for multiple minipreps than I have, Bernard.
> Nowadays, if I can't find my clone in one pass, I go right to colony hybs.
> Usually I find that something odd has happened, such as small inserts that
> give blue colonies anyway.
> I used to try a few rounds of screening with minis, then after I'd find the
> clone with colony hybs, I'd think: that wasn't so hard, why didn't I do that
> in the first place? 
>  _______________________________________________
> | Nick Theodorakis                              |


I'm in a lab of radiation-phobes/ecologically minded people
and so the amount of stress from innumerable screenings is
far less than from using a few microCi of 32P.
     I've played with DIG/alkaline phosphatase Southerns
in a previous lab with good success.  Does this work well
for colony hybridisation or does anyone recommend any other
non-rad methods for this?
     Thanks,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA