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MOLECULAR BIOLOGY: WORKING WITH DNA

NUCLEIC ACID PROBES

LABELING OLIGONUCLEOTIDES USING T4 POLYNUCLEOTIDE KINASE

Labeling Oligonucleotides Using T4 Polynucleotide Kinase Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Donald Rio at the University of California, Berkeley
Procedure Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. Add the following to a microcentrifuge tube:    2 μl of 10X Kinase Buffer    3 μl of γ-[32P]-ATP (7000 Ci/mmole or 167 μCi/ul) (CAUTION! See Hint #1)    1 μl of BSA    1 μl of Oligonucleotide Primer (see Hint #2)    13 μl of ddH2O    Add 30 Units of T4 Polynucleotide Kinase (USB) 2. Incubate at 37°C for 60 min. 3. Add 80 μl of TE Buffer. 4. Incubate for 5 min at 65°C. 5. Poke two holes in the bottom of a 0.5 ml microcentrifuge tube with a 25-gauge needle. 6. Add approximately 1 mm of glass beads to hold the resin in the tube. 7. Add approximately 150 μl of G10 resin (see Hint #3). 8. Place the 0.5 ml microcentrifuge containing glass beads and G10 resin inside of a 1.5 ml microcentrifuge tube. 9. Centrifuge for 1.5 min at setting "6" in a clinical centrifuge. 10. Discard the liquid from the bottom of the 1.5 ml microcentrifuge tube. 11. Repeat the centrifugation step and discard the liquid from the bottom of the 1.5 ml centrifuge tube. 12. Load the DNA sample onto the resin. 13. Centrifuge for 3 min at setting "6" in a clinical centrifuge. 14. Appropriately discard the column containing the nucleotides. 15. The labeled probe is in the eluate in the bottom of the 1.5 ml microcentrifuge tube. 16. Store at -20°C.
Solutions Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
0.5 M DTT 100 mM Spermidine 1 M MgCl2 2 M Tris, pH 7.6 Oligonucleotide Primer    50 ng/ul of Oligonucleotide Primer Requires between 40 to 50 ng oligonucleotide per reaction (per 1 ul) TE Buffer    10 mM Tris 1 mM EDTA pH 8.0 BSA    2 mg/ml Bovine Serum Albumin Kinase Buffer (10X)    Prepare just before use 10 μl of 1 M MgCl2 10 μl of 100 mM Spermidine 30 μl of 0.5 M DTT 17 μl of ddH2O 33 μl of 2 M Tris, pH 7.6
BioReagents and Chemicals Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
Tris Polynucleotide Kinase, T4 I3-[32P]-ATP DTT Bovine Serum Albumin (BSA) Oligonucleotide Spermidine EDTA Magnesium Chloride
Protocol Hints Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. Design the oligonucleotide primer so that it will amplify the sequence of interest. Confirm that the primer 5' end is deprotected. 3. A convenient way to measure the amount of G10 resin is to fill the top of a 0.5 ml microcentrifuge tube and add that to the microcentrifuge tube containing the glass beads.