Molecular Techniques and Methods

3'-end Labeling of DNA with Klenow Fragment

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Klenow 5 x Buffer (1 ml)
0.25 M Tris-HCl (pH 7.2) ---------------------- 250 ul of 1 M Tris-HCl
50 mM MgSO4 --------------------------------- 50 ul of 1 M MgSO4
0.5 mM DTT ------------------------------------ 5 ul of 0.1 M DTT
Distilled H2O ------------------------------------ 695 ul



2mM unlabeled dNTPs without dCTP

[a- 32P]dNTP (400 Ci/mmole)

TE-saturated Phenol: Chloroform: IAA (25:24:1)

3 M Sodium acetate (pH 7.0)

10 ug/ul Glycogen

100% Ethanol

TE buffer




PROCEDURES

1. Digest the DNA to be labelled with restriction enzyme to generate recessed 3'-ends.

2. Mix the following in the microfuge tube:

Digested DNA to be labelled
1 ug
5 x Klenow Buffer
10 ul
2mM unlabeled dNTPs without dCTP
1 ul
[a-32P]dCTP (400 Ci/ mmole, l mCi/ ml)
2 ul
Klenow fragment
1-5 u
Add distilled H2O to make a final volume of
50 ul


3. Incubate at room temperature for 10 minutes.

4. Add 1 volume of TE-saturated phenol: chloroform: IAA.

5. Vortex for 1 minute.

6. Centrifuge at 13,000g for 5 minutes.

7. Transfer the upper aqueous layer to a new tube.

8. Add 0.1 volume of 3 M Sodium acetate (pH 7.0) and 10 ug of glycogen as a career.

9. Add 2.5 volumes of 100% ethanol.

10. Centrifuge at 13,000g for 20 minutes.

11. Discard the supernatant to the radioactive waste container.

12. Redissolve the DNA pellet in 50ul of TE buffer.

NOTES



KIT INFORMATION



REFERENCES


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