Blunt end ligation problem

John R. McQuiston zje8 at cdc.gov
Wed Dec 16 08:22:05 EST 1998


I have heard that a exonuclease coprecipitates with sma I and some other 
enzymes so some sources of sma I may be contaminated with this.  I also don't 
like CIP because it seems to greatly decrease transformants regardless, for 
whatever reason.  Can you try using EcoRV or HincII as the blunt cutter?  Also 
try cutting a Blue/White vector with EcoRI, filling in and ligating.  Then 
check the Blue to White cfu ratio to see how many filled in.  A complete fill 
in should cause a frame shift and hence a white colony.  
If possible could you add a EcoRI linker into the smaI site then you have 
sticky ends?  
Any way you put it blunt end ligations stink.  

John




In article <757lm3$ck5$1 at nuscc.nus.edu.sg>, scip7290 at leonis.nus.edu.sg says...
>
>Dear all,
>
>        I am currently facing a problem with ligating Klenow treated
>insert into a CIP-SmaI vector.  I have cloned two fragments into this
>vector before, both unidirectional sing two different enzymes.  Then I
>tried to clone in an EcoRI insert, so I Klenow treat, and CIP my vector.
>No transformants obtained.  I did the following trouble shooting :
>1. Change ligase (2 different ligase)
>2. Change competent cells (check competency with the vector itself)
>3. Tried religation of vector.
>
>        When I tried religating the vector, I could only get a handful of
>transformants.  I know that this should not be the case, but I do not know
>where else could be the problem.  I changed the water, competent cells,
>ligation conditions etc.  I feel that if I cannot religate, something must
>be terribly wrong.  Am I missing something important here I-(  I refered
>to Sambrooks etc  Could someone please just send me a tried and tested
>protocol for blunt end ligation.  I will be very thankful.
>
>HS     




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