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Fill-in Labeling of DNA : directional end label

点击： 作者：51protocol收集 来源： 时间： 2007-03-29 　本站论坛

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

ml) 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 MgCl

ml 1 M Tris 7.52 100 ml 1 M MgCl2

10 mM DTT 100

0.5 mg/ml BSA 50

250

ml 0.1 M DTTml 10 mg/ml BSAml Qstore at -20° in 50 ml aliquotes

dNTP Mix

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

ProcedureDigest 2

•

• Resuspend the pellet in 19

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.

ml Qml each of 3 cold dNTP's (10 mM stocks, see above)a-32P-dATP with EcoRI Labeling).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.ml Q, then add:ml dNTP mixml 10X Klenow Bufferml a 32P dNTPml Klenowml Q and digest with the second enzyme for 30' at 37 degrees.ml Q.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.

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