Problems with one base extension cloning.

Alan KAY kay at lyon151.inserm.fr
Wed Jul 10 10:28:25 EST 1996


Hi,
I am having problems cloning fragments cut with Tth111 I. This enzyme cuts 
the sequence GACN/NNGTC, so it leaves a one base 5' extension. I know that 
such ends are difficult to ligate, but I have done it before, and Tth111 I is 
the worst I have seen. The N of the 5' extension is not the problem since I 
am re-inserting mutated fragments back into the wild-type construct and so 
the donor and acceptor overhangs are compatible. I am doing PCR mutagenesis, 
and Tth111 I is the only convenient unique site available.

Does anyone have hints for one base overhang ligations? Are tricks that are 
used for blunt-end ligations, i.e. PEG or low ATP concentrations, also 
applicable to one base overhangs?

Thanks for any help.

Alan KAY




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