Spin Column chromatography
Used to removed unincorporated nucleotides from labelling reactions.
- Prepare Sephadex G-50 (medium) by adding appropriate amount of dry beads to 100 ml TE buffer such that the beads will swell to 50 ml. Autoclave.
1. Rock bottle gently to make a uniform slurry.
2. Place a disposable 3ml chromatography column into a 15 ml round bottom tube. Add 2 ml of the slurry to the column.
3. Spin the tube in swing out cups of the Jouan centrifuge, using the pre-programmed setting (to determine the right time and speed on a different machine, use isotope to see how long and how fast to spin without letting the free dCTP go through the column to a significant degree. Co-load some DNA to ensure that this indeed flows through under new conditions. We go about 1500rpm for about 90 seconds)
4. Dump out flow-through. Put a 1.5ml microfuge tube with a cut-off cap into the bottom of the tube.
5. Replace the column and add your 200 ul sample to the center of the top of the bead bed.
6. Spin on the same program.
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Labeling oligonucle