Fill-in Labeling of DNA : directional end label

http://axon.med.harvard.edu/~cepko/protocol/mike/F1.html

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 m l) aliquotes at -80 degrees and thaw on ice just prior to use

10 X Klenow Buffer

0.5 M Tris 7.5 500

0.1 M MgCl₂

m l 1 M Tris 7.5 2 100 m l 1 M MgCl₂

10 mM DTT 100

0.5 mg/ml BSA 50

250ml 0.1 M DTT m l 10 mg/ml BSA m l Q store at -20℃ in 50 m l aliquotes

dNTP Mix

21

3

Note: leave out the dNTP which will be used for labeling of the chosen restriction site (i.e.

Procedure Digest 2

Resuspend the pellet in 19

25

5

5

1

Incubate at room temperature for 30'.

Phenol/chloroform extract and EtOH ppt. Resuspend in 16

 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

Count 1

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.

m l Q m l each of 3 cold dNTP's (10 mM stocks, see above) a - 32 P-dATP with EcoRI Labeling). m g 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. m l Q, then add: m l dNTP mix m l 10X Klenow Buffer m l a 32 P dNTP m l Klenow m l Q and digest with the second enzyme for 30' at 37 degrees. m l Q. m l 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.