Tuesday, October 21, 2003

Description
Labeling Oligonucleotides


Procedure
1. Combine the following reactants in a microcentrifuge tube:
0.5 靗 of Denaturing Buffer
50 ng of the Oligonucleotide to be labeled (in TE Buffer)
Bring the total reaction volume to 5 靗 with ddH2O

2. Incubate the reaction at 70癈 for 5 min.

3. Place the reaction on ice and centrifuge briefly in a microcentrifuge to remove the condensate from the tube lid.

4. Add the following items to the reaction tube in the listed order:
1.25 靗 10X Kinase Buffer
0.5 靗 100 mM DTT
5 靗 of 10 uCi/ul ?[32P]ATP (CAUTION! See Hint #1)
0.75 靗 (7.5 Units) of T4 Polynucleotide Kinase

5. Incubate the reaction at 37癈 for 1 hr.

6. Add the following items to the reaction tube:
36 靗 of TE Buffer
50 靗 of 4.0 M Ammonium Acetate
1 靗 of 10 mg/ml tRNA
200 靗 of 100% Ethanol
Mix the contents by vortexing

7. Centrifuge the tube in a microcentrifuge at full speed for 10 min to pellet the DNA.

8. Remove the supernatant and resuspend the pellet in 100 靗 TE Buffer and count 1 靗 in Scintillation Fluid in a Scintillation Counter (see Hint #2).


Recipes
tRNA (10 mg/ml)

4.0 M Ammonium Acetate

TE Buffer 10 mM Tris
1 mM EDTA
pH 8.0


?[32P]ATP (10 霤i/靗)

100 mM DTT

Kinase Buffer (10X) 50% (v/v) Glycerol
100 mM MgCl2
500 mM Tris, pH 9.5


Denaturing Buffer 200 mM Tris, pH 9.5
1 mM EDTA
10 mM Spermidine



Supplies


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

2. The DNA pellet need not be washed with 70% Ethanol.