Labeling Oligonucleotides

Tuesday, October 21, 2003

Description
Labeling Oligonucleotides

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

2. Incubate the reaction at 70C 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 l 10X Kinase Buffer
0.5 l 100 mM DTT
5 l of 10 uCi/ul -[32P]ATP (CAUTION! See Hint #1)
0.75 l (7.5 Units) of T4 Polynucleotide Kinase

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

6. Add the following items to the reaction tube:
36 l of TE Buffer
50 l of 4.0 M Ammonium Acetate
1 l of 10 mg/ml tRNA
200 l 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 l TE Buffer and count 1 l 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 Ci/ l)

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.

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Labeling Oligonucleotides