Removal of Unlabelled Probe DNA (Spin Column Procedure)

Removal of Unlabelled Probe DNA (Spin Column Procedure)

  1. To prepare the spin columns, in a 12X75 mm disposable tube (use 15 ml PPT), place 1000 µl blue pipet tip with a 1/4-inch bevel cut in the end. With a second blue tip, cut approximately 1/8 inch off of the end and pack appro. 1/4 inch of sterile glass wool in the bottom of the cut end. Insert the sterile glass wool tip into the first tip (See the figure in detail).
  2. With a 10 ml pipet, fill the glass wool tip with hydrated Sephadex G-50 all the way to the top. To prevent bubbles while filling, let the sephadex run down the side of the tube. (If desired, centrifuge 30 sec to 1 min at top speed). All the buffer to drain out of the tip and add more sephadex to round over the top of the tube.
  3. Add 20 µl of Salmon Sperm DNA (does not have to be denatured) to the top of the column.
  4. Spin the column for 4 min. at 1,000 rpm (250 g).
  5. After removing the dried sephadex blue tips, pour out the the buffer from the bottom of the tube into a MFT. Flip the buffer out of the bottom tip into the same MFT.
  6. Reassemble the tops and place them back in the tube.
  7. Add the 32P probe (after adding RSB) to the top of the column.
  8. Rinse the MFT with 100 µl STE and transfer to the column.
  9. Spin the column again for 4 min at 1000 rpm (250 g).
  10. Estimate the percent of incorporation of 32P into a probe: After the last centrifugation spin, hold the upper part of the spin column about one inch from the photomultiplier tube on the Geiger Counter and measure. Measure the tube containing the purified probe in the same fashion as the tip. If the percent incorporation is 50 %, both the tip and the probe should be equal. If 25 % of the 32P in the tip and 75 % in the probe, calculate the difference. A good oligo labeling is somewhere between 60 to 80 % incorporation.

Hydration of Sephadex G-50

To hydrate the sephadex G-50 beads, weight out 30 g and add appr. 400 ml of STE. Autoclave, and let sit overnight at 4 C. The next day, pour off the aqueous layer of buffer and add 100 to 200 ml of new STE. Store at 4 C. Just prior to use, mix the buffer and sephadex by inverting several times.

Sterilization of glass wool

Use scissors, gloves, lab coat, a face mask. Glass wool irritate the skin and lungs.

Fill a 250 ml glass beaker with glass wool. Cover beaker with aluminium foil and autoclave for 45 min on dry cycle.