Protocol R.1

Random Prime Labeling of DNA

Solutions

Buffer A

1.25 M Tris 8.0 625 ml 2M Tris 8.0

0.125 M MgCl2 125 ml 1M MgCl2

0.5 mM dGTP 5 ml 100 mM dGTP

0.5 mM dTTP 5 ml 100 mM dTTP

222 ml Q

18 ml bME

 

Buffer B

2 M HEPES pH 6.6

 

Buffer C

135 ml Random Hexamer Mix

165 ml TE

 

Random Hexamer Mix (10 mg/ml)

50 OD units random hexamer (Pharmacia 27-2166-01)

250 ml Q

ABC Buffer

10 ml Buffer A

25 ml Buffer B

15 ml Buffer C

 

 

 

 

 

Procedure

• Boil 1 ml (0.5 mg) probe in 11 ml Q for 5 minutes and immediately place on ice for 5 minutes.

• Add the following: 24 ml ABC Buffer

4 ml BSA (10 mg/ml)

1 ml Klenow

5 ml a32P dCTP

5 ml a32P dATP

Incubate at room temperature for 1 hour.

• Phenol/chloroform extract and ethanol precipitate with 0.5 volumes NH4OAc.

• Wash with 80% EtOH and dry. Remove unincorporated nucleotides with a NENSORB column (see Protocol S.5).