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 8.0 2 125 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 dGTP ml 100 mM dTTP ml Q ml b ME ml Random Hexamer Mix ml TE ml Q ml Buffer A ml Buffer B ml Buffer C ml (0.5 m g)probe in 11 ml Q for 5 minutes and immediately place on ice for 5 minutes.ml ABC Buffer ml BSA (10 mg/ml)ml Klenow ml a 32 P dCTP ml a 32 P dATP4OAc.

0.125 M MgCl₂