RADIOLABELING OF PROBES FOR ELECTROPHORETIC MOBILITY SHIFT ASSAYS

 

Materials:

                        g-32P 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

                        32P waste nearby

 

Procedure:

 

  1. 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.

 

  1. 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.

 

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

 

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

 

  1. Add 1 ul of T4 kinase.

 

  1. Close the tube, flick gently to mix

 

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

 

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

 

  1. 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 column.

 

  1. 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.)

 

  1. Unscrew the syringe, remove the barrel.  Add 75 ul of fresh STE to the top of the column and repeat step 10 above.

 

  1. 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.

 

  1. 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.

 

  1. 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.

 

  1. 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.

 

  1. Place the original tube and the dilution in the freezer.