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v) 10-50 fold dilution of the probe
vi) NE usually 2-4
• Incubate at room temperature for 30 minutes and load directly onto gel with no loading dye. Run the gel at 200V for 4 hours. Note: it is best to pre-run the gel during the 30 min binding reaction.
ml Q with 1 ml 10X Annealing buffer. Place in a 65° water bath for 2 min and cool in 50 ml of the 65° water in a beaker on ice. This takes 15-20 minutes.ml of the annealed oligo mixtureml 10X Klenow bufferml dNTP mix (21 ml Q, 3 ml 10 mM dATP/dTTPml a32P dCTPml Qml Klenowml TE and count. I usually get 200,000-400,000 cpm per ml.ml 10% APS, 180 ml TEMEDml 2X Binding Bufferml dI/dCml (see table)mg is sufficient
mg/ml stock and store in 100 ul aliq. at -80° C
10 mM EDTA 8.0 20
500 mM NaCl 100
280
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