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
c) cut with the desired enzymes
d) gel purify the desired product (monomer) and ligate it
Oblivion together does not frighten me, beloved.
-- Thalassa (in Anne Mulhall's body), "Return to Tomorrow",