HELP: ligation (used to be Re: cip & geneclean)

coyne at uctvax.uct.ac.za coyne at uctvax.uct.ac.za
Thu Aug 13 03:00:10 EST 1992


Dear Bruce and fellow netters,

We were very pleased to see the following posting by Bruce Roe as we are
currently experiencing similar problems to those reported by Michael; except
that we are trying to clone 10 kb gel purified, BamHI chromosomal DNA
fragments into BamHI cut and cipped Bluescript.  Although Bruce's suggestions
are extremely helpful, I have a question relating to point #3 below:

In article <10AUG199208480766 at aardvark.ucs.uoknor.edu>, broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:
> In article <1992Aug9.912.1309 at channel1>, "michael coyne" <michael.coyne at channel1.com> writes...
>>Hello fellow molbio.metters:
>> 
>>I have a question to which I have been unable to get a clear answer.  I
>>do not expect a cut-and-dried answer, but perhaps a consensus would be
>>helpful.
>> 
>>We have been attempting to clone a fragment of DNA into a high
>>copy-number vector (a derivative of the pUC plasmids).  We have had some
>>problems with incomplete digestion of the vector causing high
>                ^^^^^^^^^^^^^^^^^^^^
>>background, thus making identification of recombinant clones difficult
>>(using lacZ blue/white identification).  
> 
> Suggestions:
> 	1. Try a series of simple control ligation experiments where
> 	   you linearize the vector with the restriction enzyme for
> 	   various times at various enzyme/vector rations.  Then just
> 	   do a transformation without the ligation step.  This will
> 	   help you determine the optimum digestion conditions to
> 	   minimize any un-cut vector.
> 	2. Once you've found digestion conditions where there is minimum
> 	   un-cut vector, then do a time study for BAP or CIP
> 	   de-phosphorylation.  (usually need 30min, 1hr, 2 hr)
> 	   Do a ligation/transformation for each time point without
> 	   added insert to see the efficiency of the phosphatase.
> 	3. Repeat the ligation/transformation using the de-phosphorylated
> 	   vector with minimal background produced in step #2 and this
> 	   time use varying amounts of insert for the ligation, holding
> 	   the vector amount constant.

QUESTION:	What is the recommended starting concentration of total DNA 
(or that of insert & vector)?  We have been starting with 5 pM of total DNA and
then performing 2-fold serial dilutions of the insert DNA while keeping the
concentraion of vector DNA constant.  However, we are continually plagued with
low numbers of transformants (and inserts) following transformation of E. coli
LK111 competent cells.

(rest of Bruce's original posting deleted; still available on
molbiol.meths-reagnts)

Thank you (in advance) for any assistance with our problem,
Vernon Coyne
coyne at uctvax.uct.ac.za

  



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