Purification of radiolabeled oligonucleotides by CPB precipitation assay
This protocol describes the separation of radiolabeled oligonucleotides from unadulterated radiolabeled material by quantitative differential precipitation with a cationic detergent, cetylpyridinium bromide (CPB). This method was originally used to recover DNA eluted from hydroxyapatite columns in phosphate buffer (Geek and Nasz 1983), and can also be used to precipitate radiolabeled nucleic acids including oligonucleotides. This experiment was derived from Molecular Cloning Laboratory Guide (Third Edition), Previous Edition, by Peitang Huang.
Operation method
Experiments on purification of radiolabeled oligonucleotides by CPB precipitation method
Materials and Instruments
Cetylpyridinium bromide EDTA-Tris EDTA-Tris-DNA solution Ethanol-sodium acetate solution Ethanol-sodium acetate TE (pH 7.6) or appropriate prehybridization solution Raw materials for nucleic acid and oligonucleotide purification Move makings For more product details, please visit Aladdin Scientific website.
Dry ice Ethanol bath
Solutions and buffers
Dilute the storage solution to the appropriate concentration.
Cetylpyridinium bromide (CPB) 1%, m/V)
Dissolve 1 g of CPBW (sigma) in 100 ml of water; heating and stirring the solution with a magnetic stirrer accelerates dissolution. After all the detergent was dissolved, the solution was cooled to room temperature and filtered through nitrocellulose filter membrane (0.45um pore size).
EDTA-Tris(0.5mol/L,pH6.0)
Dissolve 0.1 mol EDTA in 60 ml of water and while stirring the solution, add Tris base (powder) until the solution pH reaches 6.0, at which point the concentration of Tris is about 1.2 mol/L. Add water to make the volume of the solution 200 mL.This is different from the conventional method of preparation but is necessary for the cationic detergent to precipitate nucleic acids efficiently (Sibatani 1970). Sibatani1970).
EDTA-Tris-DNA solution
Dissolve vector DNA (salmon DNA) with 50 mmol/LEDTA-Tris (pH 6.0) (1:10 dilution of 0.5 mol/LEDTA-Tris solution) to a concentration of 50ug/ml Vector DNA should be homogeneous in size and should not interfere with hybridization of radiolabeled nucleic acids to target DNA. Sonication or salmon DNA fragments will satisfy the requirements of most experiments (sonicated salmon DNA is 600-5000 bp in length and can be purchased from Pharmacia or prepared according to Scheme 10 in Chapter 6).
Ethanol-sodium acetate solution
80% (V/V) ethanol
20%(V/V) Sodium acetate (0.1mol/L, pH5.2)
TE (pH 7.6) or appropriate prehybridization solution
See step 8.
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 at 68°C.
Specialized equipment
Dry ice/ethanol bath
method
1. Add 5-10 times the volume of EDTA-Tris-DNA solution to the microcentrifuge tube containing the radiolabeled oligonucleotide and mix thoroughly.
For efficient precipitation, it is important that the ionic component of the EDTA-Tris-DNA solution predominates in the final reaction mixture, and that the final volume of the mixture and the concentration of the radiolabeled oligonucleotide do not affect the efficiency of the method.
2. Add sufficient 1% CPB to bring the decontaminant concentration of the mixture in the centrifuge tube to 0.1% and mix well.
3. Place the tube in a dry ice/ethanol bath until the mixture freezes. Remove the tube from the ice bath and allow the mixture to thaw at room temperature.
4. Centrifuge the tubes at maximum speed for 5 min at 4°C and carefully remove the supernatant from the tubes using a Pasteur pipette or a micropipette fitted with a disposable blue 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 radioactive material, pipette tips, and centrifuge tubes should be handled with care.
5. Add 500 ul of distilled water to the tube, shake for 20 s, and centrifuge again as in step 4.
6. Pipet the supernatant from the tube, add 500ul of ethanol-sodium acetate solution, shake for 15 s, and centrifuge at maximum speed for 2 min at room temperature.
7. Repeat step 6.
8. Carefully remove the supernatant. Open the cap of the centrifuge tube and place it behind a test bench polyisobutylene acid resin Plexiglas protective screen until the remaining visible ethanol evaporates cleanly. Dissolve the precipitated oligonucleotide with 20 to 50 ul of TE (pH 7.6). If radiolabeled oligonucleotides are used as probes, dissolve in a small volume of prehybridization solution.