Removal of 5' overhang

Arnoud van Vliet avvliet at knoware.nl
Fri Nov 6 18:37:20 EST 1998


Hi Neil

relatively simple: use the exonuclease properties of E. coli DNA polymerase
I or T4 polymerase. I use DNA pol I (not Klenow!):

exonuclease step; mix :
DNA
low-salt restriction buffer
DNA polI

Incubate 15 min at 37 degrees C.

Add dNTP nucleotide mix to normal concentrations (about 100 uM) and incubate
an additional 15 min (polymerase step), and then either gel-purify or
fenol/chloroform extract followed by ethanol precipitation.

Hope this helps,
Arnoud

Fraser Dr N J wrote in message <36432249.167E at ggr.co.uk>...
>Probably a simple question, but can't think of the answer at the moment.
>How do I remove a 5' overhang ? I want to destroy an NcoI site (CCATGG)
>to remove the ATG so simple filling in won't do.
>Cheers - Neil





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