Experimental purification of radiolabeled oligonucleotides by size exclusion chromatography
Experimental purification of radiolabeled oligonucleotides by size exclusion chromatography
When radiolabeled oligonucleotides are used in enzymatic reactions such as primer extension reactions, it is necessary to completely remove the unadulterated radiolabel. This protocol describes a method for separating radiolabeled oligonucleotides from unadulterated radiolabelers by using the difference in the rate of movement of oligonucleotides and mononucleotides during size exclusion chromatography. This experiment was derived from Molecular Cloning Laboratory Guide (Third Edition), Upper Volume, by Peitang Huang.
Operation method
Experimental purification of radiolabeled oligonucleotides by size exclusion chromatography
Materials and Instruments
Chloroform EDTA Ethanol Phenol: Chloroform Sodium Acetate TE Tris-Cl Tris-SDS Chromatography Buffer Radiolabeled Oligonucleotides Raw Materials for Purification Move makings For more product details, please visit Aladdin Scientific website.
Gel filtration resin Glazed cotton Pasteur pipette
Solutions and buffers
Dilute the storage solution to the appropriate concentration.
Chloroform
Optional, see step 7.
EDTA(0.5mol/L,pH8.0)
Ethanol
Phenol: chloroform
Optional, see step 7.
Sodium acetate (3 mol/L, pH 5.2)
Optional, see step 7.
TE (pH 7.6)
Tris-Cl (1mol/L pH8.0)
Optional, see step 7.
Tris-SDS Chromatography Buffer
10 mmol/L Tris-Cl (pH 8.0)
0.1% (m/V) SDS
Nucleic acids and oligonucleotides
Radiolabeled oligonucleotides
The purified material is the reaction mixture of Scheme 2 (step 3 or step 5), and the T4 phage polynucleotide kinase has been inactivated at 68°C. The purified material is the reaction mixture of Scheme 2 (step 3 or step 5).
Specialized equipment
Gel filtration resin (Bio-Gel P-60 fine-grade or Sephadex G-15)
Bio-Gel P-60 (fine-grade) is available from Bio-Rad, and Sephadex G-15 is available from most chemical suppliers (e.g., Sigma) Bio-GelP-60 is a pre-swollen product, while Sephadex G-15 must be swollen and equilibrated before use.
Broken Glass Cotton
Wrap a small amount of glass wool in aluminum foil and autoclave microcentrifuge tubes (1.5 ml) at 15 psi (1.05 kg/cm2 ) according to the sterilization procedure for the wrapper in a tube rack or collector for collection of radiolabeled oligonucleotide chromatography eluates.
Pasteurized Pipettes
Additional Reagents
The reagents needed for Step 6 are listed in Scheme 7 in Chapter 13.
Methods
1. Add 30ul of 20 mmol/L EDTA (pH 8.0) solution to a microcentrifuge tube containing radiolabeled oligonucleotides. Store samples at 0°C while preparing the size exclusion resin.
For convenience, Bio-GelP-60 resin is used as an example in this protocol and can be used equally effectively with SepbadexG-15.
2. Prepare the Bio-GelP-60 column using sterilized Pasteur tubing.
a. Equilibrate Bio-Gel P-60 resin supplied by the manufacturer with 10 times the volume of Tris-SDS Chromatography Buffer. b. Equilibrate Bio-Gel P-60 resin with Tris-SDS Chromatography Buffer.
If a centrifugal evaporator (Savant SpeedVac or equivalent) is available, the Bio-Gel P-60 column can be loaded and rinsed with 0.1% ammonium bicarbonate solution and the collected radiolabeled oligonucleotide fractions can be dried in the centrifugal evaporator (Step 6) without the need for extraction of organic solvents or ethanol precipitation of the oligonucleotides.
b. Stuff a ball of sterilized glass wool into the bottom of a sterilized Pasteur pipette.
A glass capillary tube also makes a good stuffing tool. ...
c. After preparing the tubing, pour in a small amount of Tris-SDS Chromatography Buffer and check the flow rate of the buffer (one drop per second).
d. Pour the Bio-GelP-60 Resin Slurry into the tubing. The resin sinks with gravity, the buffer flows out, and the column forms rapidly. Add more resin slurry so that the glass wool plug to the constriction near the tip of the pipette is completely filled.
e. Rinse the column with 3 ml of Tris-SDS Chromatography Buffer.
It is important not to let the chromatography tubing run dry. Seal the bottom of the tubing with sealing film if necessary.
3. Pipet the excess buffer from the top end of the resin and quickly add the radiolabeled oligonucleotides to the resin.
(volume 100ul or slightly less).
4. 100ul of buffer is added immediately after the sample enters the chromatography resin. After the buffer enters the resin, new buffer is replenished successively without letting the column run dry.
5. Detect the flow of radiolabeled oligonucleotides with a handheld microprobe. When the effluent begins to show radioactivity, collect two drops of liquid in a microcentrifuge tube.
WARNING: Phosphorylation reactions often use >100uCi radiolabeled ATP, so the dose of radioactivity in the supernatant is significant, and unadulterated radiolabel, pipette tips, and microcentrifuge tubes should be handled with care and caution.
6. When the radioactive liquid is nearly completely out, measure the Cherenkov counts of the components with a liquid flash meter (see Appendix 8). If the fast-moving elution peaks (radiolabeled oligonucleotides) can be clearly separated from the slower-moving unadulterated [ γ-32P ]ATP, all fractions containing radiolabeled oligonucleotides can be combined. If the peaks are not clearly separated, take about 0.5 ul of each fraction and analyze it by determining the radioactivity bound to a DE-81 filter membrane (see Step 3 of Option 7 in Chapter 13 for details of the method) or by thin-layer chromatography. The radiolabeled oligonucleotide fractions that do not contain undoped radioactivity [ γ-32P ] are then combined.
7. If the radiolabeled oligonucleotide is to be used in an enzymatic reaction, proceed as follows. Otherwise, proceed to step 8.
a. Extract the combined labeled oligonucleotide fractions with an equal volume of phenol-chloroform.
b. Reverse the organic phase with 50ul of 10 mml/L Tris-Cl (pH 8.0) and combine the two aqueous phases.
c. Extract the combined aqueous phases with an equal volume of chloroform.
d. Add 0.1 volume of 3 mol/L sodium acetate (pH 5.2), mix briefly, add 3 times the volume of ethanol and allow to stand at 0°C for 30 min. Centrifuge at maximum speed for 20 min at 4°C. Remove ethanol from the tubes (very low radioactivity) using a micropipette fitted with a disposable tip.
8. Add 500 ul of 80% ethanol to the centrifuge tube, shake briefly, and centrifuge for 5 min at maximum speed.
9. Remove the ethanol from the tube using a micropipette fitted with a disposable tip. The centrifuge tube is placed, open mouthed, behind the Plexiglas screen of the test bench polyisobutyrylate resin until the remaining ethanol has evaporated.
10. Dissolve the precipitated oligonucleotides with 20ulTE (pH 7.6) and store at -20°C.