This protocol was designed to generate directionally end-labeled probes for DNaseI footprinting but it can be used for any application that requires end-labeled DNA probes. Solutions 10 mM dNTP Stocks Thaw100 mM stocks (NEB or Boehringer Mannheim) on ice and dilute 10-fold in Q. store small (10-20 ml) aliquotes at -80 degrees and thaw on ice just prior to use 10 X Klenow Buffer 0.5 M Tris 7.5 500 ml 1 M Tris 7.5 0.1 M MgCl2 100 ml 1 M MgCl2 10 mM DTT 100 ml 0.1 M DTT 0.5 mg/ml BSA 50 ml 10 mg/ml BSA 250 ml Q store at -20℃ in 50 ml aliquotes dNTP Mix 21 ml Q 3 ml each of 3 cold dNTP's (10 mM stocks, see above) Note: leave out the dNTP which will be used for labeling of the chosen restriction site (i.e. a-32P-dATP with EcoRI Labeling). Procedure • Digest 2 mg of CsCl purified plasmid DNA (Protocol C.1) with the restriction endonuclease corresponding to the end to be labeled. phenol/chloroform extract and EtOH ppt. • Resuspend the pellet in 19 ml Q, then add: 25 ml dNTP mix 5 ml 10X Klenow Buffer 5 ml a 32P dNTP 1 ml Klenow Incubate at room temperature for 30'. • Phenol/chloroform extract and EtOH ppt. Resuspend in 16 ml Q and digest with the second enzyme for 30' at 37 degrees. • Gel purify by running out the restriction digest (5 minutes 100 mA) on a 1% minigel and spin purifying (Protocol D.5). • Resuspend the purified fragment in 100 ml Q. • Count 1 ml by spotting onto Whattman 3mm filter paper and counting for 1 min. I get 20,000-50,000 cpm for restriction fragments and 200,000-400,000 cpm for double stranded oligos. Anything less than 15,000 for restriction fragments should be trashed. • Probes labeled using this protocol are usually good for 1-2 weeks but the best results are obtained when the probe is used within the first few days after labeling. |
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