Kenneth A. Johnson, Ph.D Lab | Methods

 

32P RadioLabeling Oligonucleotides for Kinetic Assays

Written by: Matthew Kellinger
Version: 2010-06-07

Purpose: for the 5' labeling of purified oligonucleotides with 32P for kinetic assays.

Assemble the labeling reaction:
  • 50µL Total volume
  • 19µL dH2O
  • 20µL of 100µM PAGE purified oligo to label (usually 25mer)
  • 5µL of 10× T4 PNK buffer
  • 3µL γ32P-ATP
  • 3µL T4 Polynucleotide Kinase (from NEB)
  • 1) Incubate the labeling reaction for 1 hour at 37°C (Keep reaction shielded at all times).
  • 2) Quench reaction by heating to 85°C for 5 minutes.
  • 3) Using the back of a razor blade, remove the TLC resin from the TLC plate to from two separate 2cm lanes. Spot 1µL of "Pre" Bio-Spin reaction 1 inch from the bottom of the plate in lane 1.
  • 4) Remove excess hot ATP by Bio-Spin 30 spin column. NOTE: Directions taped to the side of the hood in the radioactive area.
    • KEY POINTS TO SUCCESS WITH BIO-SPIN 30
    • It seems obvious, but follow the instruction exactly.
    • The water wash steps are important for buffer switching
    • The spin time for the purification (4 minutes) should be exact. Too little and the labeled oligo won't be thru the column. Too long and the excess labeled ATP will contaminate your sample.
  • 5) Dry the TLC plate spots thoroughly with the heat gun. (if they are not completely dry the "spots" will become "streaks" when the samples migrate).
  • 6) Run the TLC plate in 375mM Phosphate buffer with an 4.0pH. Stop the TLC plate before the mobile phase reaches the top of the plate.
  • 7) Dry the TLC plate with the heat gun. Wrap the plate in plastic wrap.
  • 8) Expose a previously blanked storage phosphor cassette screen for 30 seconds. Open the cassette and move the TLC plate down about 3 inches. Repeat the exposure for 1 minute.
  • 9) Repeat step 8 for 2min, 3min, and 5min. (this will ensure a proper exposé).
  • 10) Scan the Storage Phosphor screen on one of the buildings Typhoon scanners (DNA core facility or 2nd floor darkroom).
  • 11) Using ImageQuant, quantify the density of the Pre- and Post- Bio-spin samples.
  • 12) Use C1V1=C2V2 to determine the final concentration of the labeled oligo where:
    • a. C1 = is the starting concentration of the oligo in the labeling reaction (40µM if a 100µM stock oligo was used in a 50µL reaction).
    • b. V1 = is the density of the Pre Bio-Spin spot from ImageQuant.
    • c. C2 = is the unknown final concentration of oligo
    • d. V2 = is the density of the Post Bio-Spin spot from ImageQuant.
  • 13) Store the final labeled oligo in the Radioactives box in the Radioactives freezer properly labeled with Your Name, Compound, Isotope, Date, and Concentration.
  • Note: Ideally after the BioSpin Column you are left with a yield of ~80 to 100µL at 2535µM.