polish 5'overhang/cloning

Frauke Naumann fnaumann at scan.genetik.uni-koeln.de
Mon Dec 13 15:25:23 EST 1999


Hi there,
I have to remove 5' overhangs from a SalI digest. Using mung bean nuclease,
the vector is degraded (a smear all over the gel) to a great extent even
after incubation at 4°C. Cutting out the remaining band (of expected size
without degradation) of an agarose gel does not help. In subsequent
ligations I get all kind of ligation products derived from degraded vector.
Could I use a polymerase that has a 5'-3' exonuclease activity without
nucleotides? How long / at what temperature would I have to incubate so that
it doesn't eat too much of the 5' end?
Thanks for help,
Frauke





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