Digestion at the termini of PCR product?

Ashok Aiyar aiyar at biocserver.BIOC.CWRU.Edu
Sat Nov 25 10:45:58 EST 1995


On 25 Nov 1995 06:57:58 GMT, Zheng Huanquan <imazhq at leonis.nus.sg> wrote:
>I currently get a PCR product which has Nhe I at 5' terminus and BamH I 
>at 3'. I would like to ligate it into pET21a. Any one has experience in 
>digesting DNA with enzymes which sites are at termini? any protocol?

While cutting PCR fragments with enzymes close to the terminii (<6 nt),
I have done the following:
a) PCR with phosphorylated oligos using an enzyme that leaves blunt
   ends (i.e Vent, Pfu, Pwo ...)
b) self-ligate the purified PCR product for 20 minutes at 37 degrees
    This creates a multimer of the PCR product which will migrate as a 
    ladder on gels with sizes  ranging from monomer upwards .... I have 
    seen heptamers
c) cut with the desired enzymes
d) gel purify the desired product (monomer) and ligate it

Ashok

--
Ashok Aiyar
http://biocserver.bioc.cwru.edu/pp/aiyar/aiyar.html
--
Oblivion together does not frighten me, beloved.
		-- Thalassa (in Anne Mulhall's body), "Return to Tomorrow",
		   stardate 4770.3.




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