Removal of 5' overhang
hroychow at NMSU.EDU
Sun Nov 8 12:28:24 EST 1998
At 12:37 AM 11/7/98 +0100, Arnoud van Vliet wrote:
>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 :
>low-salt restriction buffer
>Incubate 15 min at 37 degrees C.
Not safe for removing single strand specifically. Mung bean nuclease should
be a better choice because of its specificity for single strand.
>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,
>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
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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