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