Radiolabeling of probes for electrophoretic mobility shift assays

 Materials:

g -32 P ATP (New England Nuclear/Perkin Elmer, CAT# NEG-035C)

“Upper strand” oligonucleotide (at 0.5 ug/ul)

“Lower strand” oliognucleotide (at 0.25 ug/ul)

T4 polynucleotide kinase (New England Biolabs)

10X T4 polynucleotide kinase buffer

 MilliQ water (distilled, deionized, nuclease-free)

STE buffer:  10 mM Tris-7.5, 1 mM EDTA, 100 mM NaCl

Stratagene “push” column

Stratagene beta shield device

Geiger counter nearby

 32 P waste nearby

Procedure:

1、Wear gloves.  Remove 32 P source vial in the lead pig from the freezer and place in the fume hood.  Let 32 P thaw at least 30 min. at room temp.

2、Assemble reaction components in an eppendorf tube as follows:

Upper strand oligonucleotide      1 ul

Water                                      7.5 ul

10 T4 kinase buffer                   1.5 ul

place the tube on a rack in the fume hood.

3、WITH GLOVES, behind a shield in the hood , remove 4 ul of 32P from the source vial and add to the eppendorf tube above.

4、Check your pipetman and gloves to make sure they are not contaminated.

5、Add 1 ul of T4 kinase.