Protocol for Trichloroacetic Acid (TCA) Precipitation
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Introduction
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., 32 P-dATP or 32 P-UTP). Then, the synthesized polymers are TCA precipitated and reaction efficiency is determined by the following formula:
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cpm per µl of sample after TCA precipitation
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cpm per µl of sample with no precipitation
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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.
Reagents and Equipment Required
Protocol
