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DNA LABELING USING PSORALEN-BIOTIN

Principle:

Psoralen-biotin intercalates into nucleic acids and upon UV irradiation, becomes covalently bonded to the nucleotide bases. Unreacted psoralen-biotin is extracted with butanol. The same protocol can be used to label RNA or oligonucleotides.

Sample:

DNA purified and linearized according to standard procedure

Reagents:

20X TE: 100 mM Tris-HCl, 10 mM EDTA, pH 8.0

Psoralen-biotin: Schleicher &Schuell # 483101, Rad-free system for probe labeling and detection.

Water saturated butanol: Add 1 mL of water to 4.5 mL butanol and mix

Materials and Equipment:

Pipettors and sterile filter tips

0.5 ml microcentrifuge tubes (siliconized)

Hot plate (boiling)

Ice bath

UV lamp

Vortex

1.0 mL fine tip transfer pipets

Procedure:

1. Resuspend the linearized DNA in 1X TE. If the DNA is in water, add 1/20th volume of 20X TE.

2. Check the DNA concentration and adjust it to 100 mg/mL.

3. Boil the DNA sample for 5 minutes to denature the DNA.

4. Rapidly cool the sample on ice.

5. Add 10 ml of the DNA to a plastic cap on ice. In the dark - Add 1ml of psoralen-biotin and mix.

6. Keep on ice and irradiate with UV lamp for 30 minutes.

7. Transfer the labeled DNA to a microtube and add 90 ml of 1X TE. Add 200 ml of water-saturated butanol and vortex for 10 seconds.

8. Centrifuge for 30 seconds at 10,000 RPM at room temperature and remove the butanol top layer. Repeat the butanol wash.

9. After the second extraction, remove the butanol completely.

10. Store the labeled DNA at -20oC. Concentration is 10 mg/ml.

References:

1. Schleicher & Schuell, Technical information for RAD-FREE System for probe labeling and detection.