Cepko/Tabin Lab ,Harvard University
http://axon.med.harvard.edu/~cepko/protocol/mike/R1.html
Protocol R.1
Random Prime Labeling of DNA
Solutions
Buffer A
1.25 M Tris 8.0 625 ml 2M Tris 8.02 125 ml 1M MgCl2
0.5 mM dGTP 5
0.5 mM dTTP 5
222
18
Buffer B
2 M HEPES pH 6.6
Buffer C
135
165
Random Hexamer Mix (10 mg/ml)
50 OD units random hexamer (Pharmacia 27-2166-01)
250
ABC Buffer
10
25
15
Procedure
• Boil 1
• Add the following: 24
4
1
5
5
Incubate at room temperature for 1 hour.
• Phenol/chloroform extract and ethanol precipitate with 0.5 volumes NH
• Wash with 80% EtOH and dry. Remove unincorporated nucleotides with a NENSORB column (see Protocol S.5).
ml 100 mM dGTPml 100 mM dTTPml Qml bMEml Random Hexamer Mixml TEml Qml Buffer Aml Buffer Bml Buffer Cml (0.5 mg) probe in 11 ml Q for 5 minutes and immediately place on ice for 5 minutes.ml ABC Bufferml BSA (10 mg/ml)ml Klenowml a32P dCTPml a32P dATP4OAc.
0.125 M MgCl
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