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Fill-in Labeling of DNA Fragments
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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Oligonucleotide Pur