Blunt end ligation problem

Low Hai Sim scip7290 at leonis.nus.edu.sg
Wed Dec 16 01:58:11 EST 1998


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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