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One of Many Ways to Use Green C-18 Resin Filled MicroColumns™ for High Throughput Purifying of Radiolabeled Oligonucleotides
1. Prepare MicroColumn for Use
a) Clean the C-18 resin thouroughly by pipetting 200ul of 100% acetonirile into a BioPak MicroColumn. Dispense and discard solution.
b) Wash away any residual acetonitrile by pipetting 200ul of ddH20 into same BioPak MicroColumn. Dispense and discard solution. Repeat 3x.
c) Compound a 10mM ammonium acetate:ddH20 solution.
d) Complete preparation of C-18 resin by pipetting 200ul of the 10mM ammonium acetate solution into the same BioPak MicroColumn. Dispense and discard.
- To maintain hydration of resin, never completely dispense solutions from the MicroColumn.
- Caution: Acetonitrile should be handled in a chemical hood, wearing gloves and goggles. Inhalation of vapors may be harmful, and contact with skin or eyes may cause irritation. Acetonitrile is also flammable. Keep away from heat and flames.
2. Bind Radiolabeled Oligonucleotide
a) If volume of the radiolabeled oligonucleotide sample is less than 200ul, bring the sample volume to 200ul with the 10mM ammonium acetate:ddH20 solution.
b) Pipette this sample into the same BioPak MicroColumn. Pipette up and down 2x.
c) Dispense and save sample solution from step 2(b) in a clean tube or well.
d) If the volume of the radiolabeled oligonucleotide sample is greater than 200ul, pipette 200ul of this sample solution into the same BioPak MicroColumn. Pipette up and down 2x.
e) Dispense and save sample solution from step 2(d) in a clean tube or well. Then, repeat steps 2(a) through 2(c) with any remaining radiolabeled oligonucleotide sample.
- Ideal volume of sample solution is 200ul.
- If quality of radiolabeled oligonucleotide in the sample solution exceeds binding capacity of the MicroColumn, Wash and Elute as shown below. Then, repeat steps 1 through 4 using the same MicroColumn.
3. Wash Radiolabeled Oligonucleotide
a) Compound a 25mM ammonium bicarbonate:ddH20 (pH 8.0) solution.
b) Pipette 200ul of this solution into the same BioPak MicroColumn. Dispense and discard.
c) Compound a 20mM ammonium bicarbonate:ddH20 solution, and a 5% acetonitrile:ddH20 solution.
d) Pipette 100ul of each of these two solutions from 3(c) into a microcentrifuge tube. Then , vortex this tube for 5 seconds to combine the solutions.
e) Pipette entire 200ul of this solution into the same BioPak MicroColumn. Dispense and discard.
f) Use previously compounded 5% acetonitrile:ddH20 solution from step 3(c).
g) Pipette 200ul of this solution into the same BioPak MicroColumn. Dispense and discard. Repeat1x.
4. Elute Radiolabeled Oligonucleotide
a) Compound a 30% acetonitrile:ddH20 solution.
b) Pipette 100ul of this solution into the same BioPak MicroColumn.
c) Pipette eluent up and down several times into an empty tube or well.
d) Dispense purified target molecule into your collecting tube or well.
e) Using the same BioPak MicroColumn, repeat steps 4(a) through 4(d).
- Addtional radiolabeled oligonucleotide may be recovered by eluting another time.
5. Dry Radiolabeled Oligonucleotide
a) Recover radiolabeled oligonucleotide be evaporating eluate to dryness in a SpeedVac for 5 minutes.
6. Redissolve Radiolabeled Oligonucleotide
a) Resuspend radiolabeled oligonucleotide in a small volume (10ul) of TE (Tris-EDTA) (pH 7.6).
Protocol adapted from Molecular Cloning, A Laboratory Manual by Maniatis, Sambrook & Frish. Modified from procedures described by Lo et al. (1984), Sanchez-Pescador & Urdea (1984), and Zoller & Smith (1984).
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