cip & geneclean

Bruce Roe broe at aardvark.ucs.uoknor.edu
Mon Aug 10 08:48:00 EST 1992


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.
	4. An alternative is to buy another batch of restriction enzyme
	   from another company and see if it cuts more efficiently.
	5. It also should be pointed out that E. coli does contain a 
	   DNA ligase which will join just linearized plasmid although
	   not as efficiently as an in vitro ligation, and thus give a
	   slight background for linear, non-de-phosphorylated vector.
	6. Finally, be sure to kill the phosphatase before the ligation
	   step and the safest, over-kill method is to phenol extract,
	   ether or chloroform wash, ethanol ppt., and wash the ppt.
	   with 70% ethanol. (although some use EDTA/heat without
	   phenol extn. etc)

> We have tried recovering the
>linearized plasmid by excising the fragment from agarose gels after
>electrophoresis, then CIP'ing the vector to minimize self-ligation, and
>ligating a mixture of the vector and our fragment.  We have so far been
>unsuccesful at recovering a clone.
> 
>This seems a pretty straight-forward experiment, and we are puzzled as
>to why it has been so far unsucessful.  While discussing the
>possibilties of why this hasn't worked (including the possibility of
>gene dosage effects due to the high copy number of the vector) one of us
>asked whether it would be better to CIP the vector digest *before*
>running it out on a gel, rather than after the ends have been
>dephosphorylated.
> 
>It may sound trivial, but is there any benefit to this?  In other words,
>is there a difference between "vector digestion - gel electrophoresis -
>recovery of fragment - CIP treatment" and "vector digestion - CIP
>treatment - gel electrophoresis - fragment recovery"?
> 
>Thanks for any comments...
> 
>Mike Coyne
>michael.coyne at channel1.com

Regarding the gel purification experiments/questions above:

	1. you should be alterted to the observation that even though
	   one thinks they are obtaining a pure, individual band on an 
	   agarose gel, the bands actually do contain a trace amount of
	   other DNA which, even though it's supposed to move faster,
	   actually tails throughout the lane.  Thus, some supercoiled 
	   plasmid will be present in your linearized band (not a lot, 
	   but sufficient to give a background in a transformation assay).
	2. gel purification step usually gives a decreased yield and
	   one needs to be concerned of insert/vector ratios in a ligation.
	3. we've actually done this experiment and see no difference
	   in if we do the CIP or BAP treatment before or after gel
	   purification.  But as mentioned above because the yield of vector
	   is lower and there is no advantage to gel purification of the
	   vector either before or after phosphatase treatment (in our hands)
	   we don't do it.

Clearly we've had similar problems, thoughts, etc. and though this summary
would be helpful to others.  There's an "oldy but goody" you may want to
read for further insights and suggestions.

	Bankier, A.T., Weston, K.M., and Barrell, B.G. (1987) Meth. Enzymol.
		 155, 51-93.

Cheers............bruce
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 \  Bruce A. Roe                     Dept. Chemistry and Biochemistry /
 /  BROE at aardvark.ucs.uoknor.edu     University of Oklahoma           \
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