Archived Protocol

<html xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns="http://www.w3.org/TR/REC-html40"> <head> <meta name=Title content="RADIOLABELING OF PROBES FOR ELECTROPHORETIC MOBILITY SHIFT ASSAYS"> <meta name=Keywords content=""> <meta http-equiv=Content-Type content="text/html; charset=macintosh"> <meta name=ProgId content=Word.Document> <meta name=Generator content="Microsoft Word 9"> <meta name=Originator content="Microsoft Word 9"> <link rel=File-List href="Radiolabeling_files/filelist.xml"> <title>RADIOLABELING OF PROBES FOR ELECTROPHORETIC MOBILITY SHIFT ASSAYS</title>  <style>  </style> </head> <body bgcolor=white lang=EN-US style='tab-interval:.5in'> <div class=Section1> RADIOLABELING OF PROBES FOR ELECTROPHORETIC MOBILITY SHIFT ASSAYS <![if !supportEmptyParas]> <![endif]><o:p></o:p> </div> <div class=Section2> 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 <![if !supportEmptyParas]> <![endif]><o:p></o:p>                         Geiger counter nearby                         32P waste nearby <![if !supportEmptyParas]> <![endif]><o:p></o:p> Procedure: <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=1 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>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.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=2 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>Assemble reaction components in an eppendorf tube as follows:</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> Upper strand oligonucleotide      1 ul Water                                                7.5 ul 10 T4 kinase buffer                         1.5 ul <![if !supportEmptyParas]> <![endif]><o:p></o:p> place the tube on a rack in the fume hood. <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=3 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>WITH GLOVES, behind a shield in the hood, remove 4 ul of 32P from the source vial and add to the eppendorf tube above.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=4 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>Check your pipetman and gloves to make sure they are not contaminated.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=5 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>Add 1 ul of T4 kinase.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=6 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>Close the tube, flick gently to mix</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=7 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>Place the tube on a rack in the 37 C incubator for 1 h.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=8 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>IN THE HOOD, behind a shield, add 60 ul of STE to the reaction.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=9 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>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.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=10 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>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.)</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=11 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>Unscrew the syringe, remove the barrel.  Add 75 ul of fresh STE to the top of the column and repeat step 10 above.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=12 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>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.</li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=13 type=1> <li class=MsoNormal style='color:red;text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>Check all components of the beta shield device to ensure that there is NO contamination.<o:p></o:p></li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <![if !supportLists]>14.  <![endif]>Add 1 ul of Lower strand oligonucleotide to the eppendorf tube, and flick gently to mix. <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=15 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>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.<o:p></o:p></li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=16 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>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.<o:p></o:p></li> </ol> <![if !supportEmptyParas]> <![endif]><o:p></o:p> <ol style='margin-top:0in' start=17 type=1> <li class=MsoNormal style='text-align:justify;mso-list:l0 level1 lfo1; tab-stops:list .5in'>Place the original tube and the dilution in the freezer.<o:p></o:p></li> </ol> </div> </body> </html>