RADIOLABELING OF PROBES FOR ELECTROPHORETIC MOBILITY SHIFT
g-32P ATP (New England
Nuclear/Perkin Elmer, CAT# NEG-035C)
strand” oligonucleotide (at 0.5 ug/ul)
strand” oliognucleotide (at 0.25 ug/ul)
polynucleotide kinase (New England Biolabs)
T4 polynucleotide kinase buffer
water (distilled, deionized, nuclease-free)
buffer: 10 mM Tris-7.5, 1 mM EDTA,
100 mM NaCl
beta shield device
- Wear gloves.
Remove 32P source vial in the lead pig from the freezer
and place in the fume hood.
Let 32P thaw at least 30 min. at room temp.
- Assemble reaction components in an eppendorf tube as
Upper strand oligonucleotide 1 ul
10 T4 kinase
the tube on a rack in the fume hood.
- WITH GLOVES, behind a shield in
the hood, remove 4 ul of 32P from the source vial and add to
the eppendorf tube above.
- Check your pipetman and gloves to make sure they are
- Add 1 ul of T4 kinase.
- Close the tube, flick gently to mix
- Place the tube on a rack in the 37 C incubator for 1
- IN THE HOOD, behind a shield, add 60 ul of STE to the
- Equilibrate a push column by adding 75 ul of STE and
forcing this through with a 10 cc disposable syringe until a drop comes
out of the bottom of the column. Mount the column on the beta
shield device, and place a clean eppendorf tube at the bottom of the
- VERY CAREFULLY, add the reaction (now in 75 ul
volume) to the top of the column bed, and gently screw on the 10 cc luer
lok syringe without the barrel.
Insert the barrel, and push the reaction mixture through the column
SLOWLY (this should take approx. 45 sec.)
- Unscrew the syringe, remove the barrel. Add 75 ul of fresh STE to the top
of the column and repeat step 10 above.
- Unscrew the syringe, remove the barrel, and repeat
step 10, but this time WITHOUT
adding any STE. This step
ensures that all the material is ejected from the column. Discard the syringe in radioactive
waste, close the eppendorf tube and ensure that approx. 100 ul of solution
is in the tube.
- Check all components of the beta shield device to
ensure that there is NO contamination.
14. Add 1 ul of Lower strand
oligonucleotide to the eppendorf tube, and flick gently to mix.
- Remove the 100 C block from the heater and place this
on your benchtop (NOT in the hood!!). Place the eppendorf tube in the
block and cover the top of the tube with a heavy object (this is to ensure
that the tube does not pop open due to the heat and splatter radioactivity
all over your bench!). Let
the tube cool to at LEAST 40 C before removing it.
- After cooling, dilute 2 ul of the sample into 200 ul
of STE. Take 2 ul of this
diluted sample and check the counts.
You want a concentration between 5-10K cpm/ul for EMSA reactions.
- Place the original tube and the dilution in the