need help for the transformation

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Wed Aug 30 04:07:36 EST 2000


In article <39AC1C32.CA42997F at biocomp.unl.edu>, Chris LaRosa
<clarosa at biocomp.unl.edu> writes
>
>> I would think this is unlikely. Usually the incompatibility regions of
>> the plasmid will stop that occurring.
>> 
>> 
>Incompatability regions..?  hmm please elaborate.. 

What I meant and said poorly was that you will have great difficulty
maintaining say different pUC19's in a single E.coli cell. If you had
selection for both a Kan derivative and a separate Amp derivative of the
same plasmid with the same origin then I believe it is possible, but in
normal circumstances you will not get this. The plasmids will segregate
so you will get some E.coli with one plasmid and some with the other. 

Bo e-mailed me that he is doing the following:


1) PCR
2)ligation and transformation
3) pick a single colony
4)mini-prep
5)ethonal precipitation for adjust the concentration
6)sequenicng

I still wonder if he should pick a single colony streak it out and
sequence some individual colonies from that. If it is a mixed culture
(but stable plasmids) in the original colony then this should get down
to single clonal types. 

Anyway just a thought.

Duncan  
  
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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