Removal of 5' overhang

Hiranya Roychowdhury hroychow at NMSU.EDU
Sun Nov 8 12:28:24 EST 1998


At 12:37 AM 11/7/98 +0100, Arnoud van Vliet wrote:
>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.


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,
>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
>
>
>
>


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




More information about the Methods mailing list