Gel Mobility Shift Assay

ed 60 ulAdd temed at last. Mix well. Now gel is ready to pour. Take 25ml pipette and try to pour the gel on the side of cast near spacers. So it trickles on the side and fills from bottom without any air bubbles trapped inside. Fill it up to the top. Take comb and insert from one side keeping angle so that each tooth of comb can be inserted one by one in Gel without trapping any air bubble. The gel should polymerize in half and hour and ready to use. Make sure its not leaking from anywhere when you leave it to polymerize. Oligoneucleotide Labeling. We want our final labeled product to be 4.8 pm/ul so dilute initial single stranded oligoneculeotide at 480 pm/ul. Now we need to anneal them.100 ul of annealing buffer ( 10mm Tris-HCl, Ph 7.5, 20mm NaCl)1 ul or each primer (480 pm of each of them)Heat it for 95* for 5 min on heat block. Leave the block at RT afterwards.Store at –20* freezer once cooled down. Labeling with 32P 2 ul of oligo 1 ul of PNK buffer 4 ul of water 1 ul T4- Kinase 2 ul of 32P ATP Incubate at 37* for 30 minutes Add 40 ul of distilled water Now we want to remove unlabelled oligos. We use Quick Spin Column (TE) for Radiolabelled DNA (Roche) for it. Keep column at room temp for half an hour. Remove the cap first and then bottomSeal to avoid generating suction. Let the supernatant drain off with gravity. Add 50 ul of your reaction.Spin them around 1000xg (2200 RPM for the machine in tissue culture room) for 4 minutes with tubes provided with column to collect labeled product. Make final volume of 100 ul by adding water. Binding reaction Now we want to initiate reaction between labeled oligos and nuclear extract of the cell line of your interest.5X GS-A bufferPoly dIdC 1ul Nuclear extract 10 ugWater to make final volume These ingredients go in each tube. Incubate on them ice for 10 min. Add cold competitor in designated tubes ( 2 ul or more according to your need)Incubate on ice for 10 min Add hot probe 1ul in designated tubes and incubate for RT for 20 min or on ice for 45 min. Add 2 ul of loading dye to stop reaction. You are ready to load them on Gel. Gel Run Before we can run the samples we need to pre-run it for 30 min at 80 volts.Prepare the buffer 0.5X TBE for running.Load the samples one by one. Making sure not to spill in upper chamber (buffer will become radioactive otherwise)Run the gel @ 120 V for 2-3 hours. When dye front is reaching lower fifth of gel. Stop the reaction. (Again if you let it long, lower chamber buffer will be radioactive). Gel drying and imaging [1] [2] 下一页
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