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.