Protocol for Trichloroacetic Acid (TCA) Precipitation

 

 

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:

cpm per µl of sample after TCA precipitation

cpm per µl of sample with no precipitation

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

• Borosilicate tubes, 12 x 75 mm (e.g., Fisher Cat #14-961-26)
• 10% TCA (e.g., Acros Cat # 42145-5000; make a 10% (w/v) solution in water) Warning: TCA is caustic!
• Carrier nucleic acid, 1 mg/ml (e.g., sheared fish sperm DNA, Ambion Cat # 9680 ; dilute to 1 mg/ml in water)
• Glass fiber filters to catch precipitate (e.g., Whatman GF/C filter circle; or Schleicher & Schuell #34 2.7-cm glass filter circles, Cat #10373828)
• Aqueous scintillation fluid (e.g., EcoLume, ICN Cat #882470)
• Vacuum manifold (e.g., Millipore Cat #XX270550)
• Scintillation vials, Scintillation counter, vortexer, pipettors

Protocol

Calculate the Fraction of Label Incorporated:

1. 7 ) by the cpm of the non-TCA precipitated sample (step 3 ) to determine the fraction of label incorporated (multiply by 100 for percent incorporation).