RADIOLABELING OF PROBES FOR ELECTROPHORETIC MOBILITY SHIFT ASSAYS

Materials:

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

“upper strand” oligonucleotide (at 0.5μg/μl)

“Lower strand” oliognucleotide (at 0.25μg/μl)

T4 polynucleotide kinase (New England Biolabs)

10X T4 polynucleotide kinase buffer

MilliQ water (distilled,deionized,nuclease-free)

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

Stratagene “push” column

Stratagene beta shield device

Geiger counter nearby

³² P waste nearby

Procedure:

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 30min.at room temp.

Assemble reaction components in an eppendorf tube as follows:

upper strand oligonucleotide 1μl

Water 7.5μl

10 T4 kinase buffer 1.5μl

place the tube on a rack in the fume hood.

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

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

Add 1μl of T4 kinase.

Close the tube,flick gently to mix

Place the tube on a rack in the 37℃ incubator for 1h.

IN THE HOOD,behind a shield,add 60μl of STE to the reaction.

Equilibrate a push column by adding 75μl of STE and forcing this through with a 10cc 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 column.

VERY CAREFuLLY,add the reaction (now in 75μl volume)to the top of the column bed,and gently screw on the 10cc 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 75μl 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.100μl of solution is in the tube.

Check all components of the beta shield device to ensure that there is NO contamination.

14.Add 1μl of Lower strand oligonucleotide to the eppendorf tube,and flick gently to mix.

Remove the 100℃ 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℃ before removing it.

After cooling,dilute 2μl of the sample into 200μl of STE.Take 2μl of this diluted sample and check the counts.You want a concentration between 5-10K cpm/μl for EMSA reactions.

Place the original tube and the dilution in the freezer.