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blunt ligation - (Jun/20/2010 )

Hi all

I am very much in trouble with the failure of blunt cloning of a 3kb insert into a 12kb vector for the past 3 months.
I have tried almost all conditions starting from temperature variation to using peg added buffers but nothing seems to work .

Can anyone please tell me how to carry on this blunt ligation ?Any method that works for blunt ligation in any lab.

Thanks in advance.


I've done quite some Blunt-end ligations. Just in short what I did:

- digest and gel-purification
- CIP the vector
- T4PNK the insert if it's a PCR product
- Ligation with negative control (only cut vector) for up to 2h on benchtop (15-20ul)

My ligations worked best if I added the insert in far excess to the ligation mixture. With the standard 3:1 ratio I didn't had any success. I used much more insert. Usually I would use as less vector as possible, from a std gel purification 2ul, just enough that I don't have any colonies on my negative plates. If the insert was low concentrated I substitute the water completely with it.