Molecular weight markers for sequencing gels prepared by depurination and cleavage of end-labelled DNA (updated 4/13/92). Light treatment of DNA with formic acid removes adenine and guanine residues with about equal frequency. Cleavage of the DNA backbone is then performed with piperidine. The DNA can be radiolabelled on either the 5' or 3' ends. 10,000 to 20,000 cpm of DNA on a gel will give a good ladder with an overnight exposure. Process at least 50,000 counts so you can run at least a few gels. The markers are stored in sequencing load buffer at -20°C. They will be good for several weeks. When used, remove and boil only the amount needed for the gel since successive boiling will reduce the quality of the ladder.

  1. Preheat the heating block in the radioactive area to 90°C.
  2. Combine radioactive DNA, 1-5 ug of sonicated salmon sperm DNA and water in a final volume of 20ul.
  3. Add 50ul of formic acid and incubate 3' at 20°C. A shorter time or slightly lower temperature (15°C) is suggested if you want the ladder to appear strong at lengths longer than 300 bases.
  4. Add 200ul of 0.3M Na Acetate pH 7.0, 20ug/ml yeast tRNA. It is important that the pH of the Na Acetate be 7 and not 5 or 6 so that the formic acid is neutralized. A lab stock of this solution may be available in the cold cabinette so ask around.
  5. Add 750ul of ethanol that is being stored at -20°C.
  6. Incubate 15' on ice and microfuge at 4°C for 15'.
  7. Remove the supernatant and check it with a Geiger counter; most of the counts should remain in the tube as a precipitate. Add 100ul of 75% ethanol (stored at -20°C), splash it around, and spin 5'. With a drawn glass pipetted or a yellow tip, remove as much of supernant as possible. Check to be certain that the counts remain in the tube. Sometimes the precipitate dislodges from the surface of the tube. If this happens, return the supernatant to the tube, spin 5', and then remove the supernatant with a drawn glass pipette.
  8. Air dry the precipitate for about 10'.
  9. Prepare a fresh 1:10 dilution of piperidine in a clean eppendorf. 100ul of this dilution will be needed for each pellet of depurinated DNA. Add the piperidine (10M) to water and thoroughly mix by triterating or capping and inverting the solution.
  10. Add 100ul of freshly diluted piperidine (now at 1M) to the precipitated DNA. Gently mix by tapping the tube and then incubate the sample at 90°C for 20' to 60'.
  11. Add 100ul of water to the sample. This is necessary for there to be a phase separation after isobutanol extraction (next step).
  12. Add 200ul of isobutanol (not stuff that has been water saturated). Mix by inverting several times and then centrifuge for 5' at 20°C. Discard the top layer by carefully removing it with a yellow tip. The radioactivity in the top layer should not exceed the first scale of the Geiger counter and can be rinsed down the drain with water.
  13. Determine the volume of the remaining aqueous layer with a pipetteman. Add 1/10 volume of 3M Na Acetate pH 6, and 2.5 volumes of ethanol (-20°C). Mix thoroughly by inverting the sample. Incubate on ice for 15'; then centrifuge for 15' at 4°C. Discard as much of the supernatant as possible and air dry the sample for 10'.
  14. Dissolve the DNA in sequencing loading buffer at 10,000 cpm/ul. Store at -20°C in a glass beaker marked as radioactive. When needed, thaw the sample and remove the desired amount. Combine this with additional loading buffer to give a volume of 4-5ul. Boil the mix for 3', quench on ice and load onto sequencing gel. Return the remaining stock of marker to the freezer. Do not subject the stock to repeated boilings.