3 & 5' Overhangs...

[L. Sumoy]

In article <2sl4t1INNp5o at medicine.wustl.edu>
HAVILAND at KIDS.WUSTL.EDU (David L. Haviland, Ph.D.) writes:
(...) I have to modify a vector in which I remove a section 
> of DNA and would like to bring the ends together.  Unfortunately, it is set 
> up using SacI and NheI both of which leave the following sticky ends:         
>                5'
>                 ---------..
>             -------------..  3' overhang left by SacI
>            3' 
> and     5'
>         ----------..
>             ------..        5' overhang left by NheI
>             3'
I would do it stepwise:
Cut with one enzyme, blunt end the ends produced by the digestion,
recut and blunt end the second set of ends. Appropriate enzyme
inactivation, phenol extraction and EtOH precipitation (or other
cleaning methods) would follow after the second restriction digestion.