Random Primed Labeling of DNA

Tuesday, October 21, 2003

Description
Random Primed Labeling of DNA

Procedure
1. Denature 30 to 50 ng of DNA by boiling for 2 min.

2. To a microcentrifuge tube add in this order:

ddH2O to give a final volume of 50 ul

10 ul of OLB

Denatured DNA from Step #1

5 ul of -[32P]dCTP (CAUTION!! See Hint #1)

2 Units Klenow Fragment

3. Incubate the reaction for 2 hr at 37C.

4. Stop the reaction by adding 200 ul of Stop Buffer.

5. Boil the reaction for between 90 sec and 3 min and add the contents directly to hybridization buffer of a Southern or Northern hybridization. There is no need to remove the unincorporated label from the reaction before adding it to the hybridization.

Recipes
Klenow Fragment

Stop Buffer 0.25% (w/v) SDS
20 mM Tris-HCl, pH 7.5
20 mM NaCl
1 uM dCTP
2 mM EDTA

OLB 2:5:3 ratio of Solution A: Solution B: Solution C

Solution C Random hexamers in TE at Absorbance280 = 90/ml (i.e. 4 mg/ml)

Solution B Adjust pH to 6.6 with NaOH
2 M HEPES

Solution A 1 ml Solution O
5 ul 100 mM dATP
5 ul 100 mM dTTP
5 ul 100 mM dGTP
18 ul 2-Mercaptoethanol

Solution O 125 mM Tris-HCl, pH 8.0
125 mM MgCl2

TE Buffer 10 mM Tris
pH 8.0
1 mM EDTA

Supplies

Tips
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

(责任编辑：泉水)

搜索

热门标签:

Random Primed Labeling of DNA