If the radiolabeled oligonucleotide is to be used only as a hybridization probe, it is generally not necessary to completely remove the unadulterated radiolabel. However, in order to minimize background, most of the unincorporated radiolabel should be separated from the radiolabeled oligonucleotide. If the oligonucleotide is longer than 18 nucleotides (this protocol), the vast majority of the unreacted radioactive precursor material can be removed by graded ethanol precipitation. This experiment was derived from Molecular Cloning Laboratory Guide (Third Edition), Upper Volume, by Peitang Huang.
Operation method
Experiments on purification of radiolabeled oligonucleotides by ethanol precipitation method
Materials and Instruments
Ammonium acetate Ethanol TE Radiolabeled oligonucleotide Raw material for purification Move makings For more product details, please visit Aladdin Scientific website.
Solutions and buffers
Dilute the storage solution to the appropriate concentration.
Ammonium acetate (1 mol/L)
Ethanol
TE (pH 7.6)
Nucleic acids and oligonucleotides
Radiolabeled Oligonucleotides
The raw material for purification is the reaction mixture of Scheme 2 (Step 3 or Step 5), and the T4 phage polynucleotide kinase has been inactivated by heating at 68°C.
Methods
1. Add 40ul of water to the tube of radiolabeled oligonucleotide, mix well, and add 240ul of 5mol/L ammonium acetate solution. After mixing again, add 750ul of ethanol pre-cooled with ice, mix well, and place at 0°C for 30 min.
Ammonium acetate was used instead of sodium acetate to ensure more efficient removal of unadulterated RNA. The solubility constants of RNA and DNA in ethanol ammonium acetate solution are greater than in ethanol sodium acetate solution. Undoped RNA was retained in the ethanol phase during the precipitation reaction.
The radiolabeled oligonucleotides were recovered by centrifugation at maximum speed for 20 min at 2.4°C. The results of the centrifugation are shown in the table below.
3. Carefully remove the supernatant from the tube with a micropipette fitted with a disposable tip.
WARNING: The supernatant contains a large portion of undoped [ γ-32P ]ATP. Phosphorylation reactions often use radiolabeled ATP greater than 100 uCi, so the dose of radioactivity in the supernatant is significant, and undoped radioactivity, pipette tips, and microcentrifuge tubes should be handled with care and caution.
4. Add 500 ul of 80% ethanol to the tube, flick the walls to wash the nucleic acid precipitate, and centrifuge again for 5 min at maximum speed.
5. Carefully remove the supernatant from the tube with a micropipette fitted with a disposable tip (the supernatant still contains a fair amount of radioactivity). Place the tube behind a polyisobutylene resin screen, open the cap, and allow the remaining ethanol to evaporate.
6.100ul of TE (pH 7.6) was used to dissolve the radiolabeled oligosiderophores.
Radiolabeled oligos can be stored at -20°C for several days. However, radiochemical damage caused by the decay of 32P can result in a decrease in the ability of the oligonucleotide to bind to the putative sequence when the shelf life is extended.