实验概要

The  efficiency of nucleotide incorporation in DNA/RNA polymerization  reactions (e.g. transcription, reverse transcription, and DNA  replication) can be determined by trichloroacetic acid (TCA)  precipitation. TCA precipitates nucleic acid polymers longer than ~20  nucleotides and can therefore be used to separate radiolabeled  nucleotides incorporated into nucleic acid from unincorporated label.

The  following is a protocol for monitoring the efficiency of radiolabeled  nucleotide incorporation in a polymerization reaction by TCA  precipitation. In the protocol, nucleic acid synthesis reactions are  carried out in the presence of a radiolabeled nucleotide (e.g., 32P-dATP  or 32P-UTP).

The  protocol has been optimized with materials from the listed vendors. The  specific details (e.g. size of tubes, amounts of carrier and sample)  are arbitrary and can be varied according to user preference.

主要试剂

Borosilicate tubes, 12 x 75 mm

10% TCA

Carrier nucleic acid, 1 mg/ml

Glass fiber filters to catch precipitate

Aqueous scintillation fluid

Vacuum manifold

主要设备

Scintillation vials, Scintillation counter, vortexer, pipettors

实验步骤

1. Dispense 198 µl of carrier DNA (1 mg/ml) into a 12 X 75 mm glass tube.

2. Add 2 µl of the nucleic acid synthesis reaction and mix thoroughly.

3. Transfer  100 µl of the diluted DNA/RNA synthesis reaction to aqueous  scintillation cocktail and count in a scintillation counter. The counts  per minute (cpm) will reflect the total amount of radiolabel present in  the reaction mixture (both unincorporated and incorporated counts).

4. Add  2 ml of cold 10% TCA (trichloroacetic acid) to the 12 X 75 mm tube  containing the remaining 100 µl diluted DNA/RNA. Mix thoroughly and  place on ice for 10 minutes. This will precipitate nucleic acids longer  than ~20 nucleotides.

5. Collect  the precipitate via vacuum filtration through a Whatmann GF/C glass  fiber filter (or equivalent). Prewet the filter with a small amount of  10% TCA prior to adding the sample.

6. Rinse  the tube twice with 1 ml of 10% TCA and then rinse once with 3-5 ml of  95% ethanol. Pass each of the rinses through the GF/C filter.

7. Place  the filter in a scintillation vial, add aqueous scintillation cocktail,  and count in a scintillation counter. The cpm will reflect the amount  of radiolabel that was incorporated.