Used to create blunt ends of either 3' or 5' overhangs by digestion of the single
stranded overhang. Advantage over S1 nuclease is its stringent specificity for single
stranded DNA -- when double stranded DNA "breathes" S1 may digest it whereas mung
bean nuclease does not. Therefore it is the nuclease of choice when exact blunting is
needed to achieve correct reading frame.
However, mung bean nuclease has some
endonuclease activity associated with it and min my experience most commercial preps
have some exonuclease activity. In general, make sure you can check the frame and
be prepared to sequence your plasmid when you are done.
10 ug of plasmid DNA cut with restriction endonuclease(s) to generate
overhangs, phenol/chloroform/ethanol precipitated, spun, washed and respun, air
Dissolve in 100 ul of buffer #1
Add 10 ul buffer #2
Incubate at 37°C for 2 minutes and add 1 ul of mung bean nuclease in buffer #3
at a conc of 4 units/ul.
Vortex and incubate at 37°C for 10 minutes
Add 1 ul 20% SDS and incubate at 65°C for 5 mins
Add 4 ul 2 M Tris pH 8.9
Add 6 ul 12 M LiCl2
Phenol/chloroform/X 2, chloroform/ethanol precipitate and wash,
spin and air dry.
6 mM NaCl
0.2 mM EDTA
300 mM NaAc pH 4.7
500 mM NaCl
10 mM ZnCl2
#2 in 50% glycerol
Mung bean nuclease (sold by PL-Pharmacia) 100 units/ul stock.