ligation of incompatible sticky ends ???

brett brett at BORCIM.WUSTL.EDU
Sun Jun 15 12:54:00 EST 1997


>In article <Pine.A41.3.95.970613200922.51726H-100000 at aix1.uottawa.ca>,
>colossus... <s535290 at aix1.uottawa.ca> wrote:
>>Here's a little stumper for you fine folks...
>>
>>The expression vector I'm cloning into will only accept NcoI/HindIII
>>inserts. It already carries a reporter gene, which you can excise by
>>NcoI/HindIII double digestion. I subsequently run such a digest, cut the
>>vector band out (9 Kb - the excised reporter is 1.7 Kb) and gel purify it.
>>
>>Ligating into this doubly cut vector is being a pain in the ass on its
>>own, but when I do a self ligation control, I manage to get a few
>>colonies. I suppose there is a possibility of carryover of uncut vector
>>during electrophoresis and gel purification...is this what's happening ? I
>>always thought I was rather careful when doing gel purification of
>>fragments and this is unnerving to say the least !!!

Here's your problem: you're overdigesting with NcoI, which seems to copurify
with some nibbling activity (NEB only recommends 5-fold overdigestion, which is
rather low). I have seen this problem myself, and easing back on NcoI, or other
nibblers, really helps. Contrary to American dogma, more is not better.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             




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