Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Mung-Bean Nuclease I 259

5' end~

3' end

IIIIIIIIIIIIIII

3' end

5' end

Fig. 1. Conversion of protruding termini to blunt ends.

3. Experimental Procedures
   for Removal of Protruding Tails
   from Double-Stranded DNA
   This technique is also known as "blunt-ending" and is performed to
   remove single-stranded"tails" left by digestion with a restriction enzyme,
   usually prior to cleavage of the DNA with another restriction enzyme. The
   following protocol is one modified from Maniatis et al. (1989) as part of
   a larger protocol for engineering a restriction site.
   3.1. Materials


4. 10X Mung-bean nuclease buffer:

• 300 mM Sodium acetate (pH 4.5)


• 500 mM Sodium chloride (NaCI)


• 10 mM Zinc chloride (ZnCI2)


• 50% Glycerol

2. 10 mM Tris-HC1 (pH 7.5).


3. Mung-bean nuclease 1.5U/I.tL.
   3.2. Methods
   For 5 lag of the DNAto be cleaved in 10 lxLof 10 mM Tris-HC1 (pH
   7.5), digest with mung-bean nuclease to generate a blunt end as follows:


4. Mix the 10 ilL of DNA with:
   10X mung-bean nuclease buffer 2 laL
   Mung-bean nuclease (1.5 U/laL) 2.5 laL
   H20 5.5 pL


5. Incubate the reaction for 1 h at 37°C.


6. Extract the reaction once with phenol:chloroform, and transfer the aque-
   ous phase to a fresh microfuge tube.