Help!!! liagtion problem

corboy at utdallas.edu corboy at utdallas.edu
Thu Jun 13 08:42:32 EST 1996


> I would try digesting your mtDNA and vector for only an hour.  O/N
> digestion would allow any exonuclease contaminants to damage your sticky
> Pst I ends.  

	I totally agree, O/N digests can cause problems especially when 
using so litte DNA.  


> You are also using way too much ligase.  I use 1.5U in each ligation with
	
	I think he is referring to the NEB ligase units, not the more 
common Weiss units.  I don't have the catalog here, but the NEB units are 
defined differently (he is using a typical amount of ligase).

> 		Do you phosphatase your vector?  If so you may be using
> too much which also has exonuclease activity.  You only need 0.1U/pmol of
> 5'P overhangs.  One final note.  You say the past 8 times you have
> attempted this you get 100s of white colonies but none of these contain
> mtDNA inserts.  How do you know this?  Have you isolated any of the
> plasmid DNA from these colonies and checked Pst I digests on gels?  If so,
> where are your inserts coming from if you started with mtDNA in the first
> place?  You may have a contamination problem.


	One question I have is regarding the ETOH ppt.  If he starts with
10ng of vector, cuts it, checks it for completion, then ups it to 500ul
and ppt's it, how can that little DNA ppt?  By then it is only 5ng/ml.  I
can't believe that will ppt without carrier.  And if it won't, where do
the control colonies come from?  
	Also, assuming that those are kosher, they could well be from
traces of uncut vector from the digest, even tho undetectable on a gel. 
This goes back to the O/N digest; 99% of the DNA gets cut, but damaged by
an exo cont; the remaining 1% uncut transforms and gives lots of colonies
in the pUC18 alone control.  GEL PURIFY, people, GEL PURIFY.  It may add a
day to your construction, but can save weeks of troubleshooting when
things come out screwy.  For example, when you try something 8 times and 
it still doesn't work (to the original poster: nothing personal, I know 
this fro mexperience).

Mike Corboy
University of Texas at Dallas

P.S.  What's the longest distance between two points?  
      A SHORTCUT
      Do it right the first time, and you won't have to figure out what 
went wrong nearly as often.




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