Lab Resources

Oligonucleotide Radiolabeling

Reaction sample: Volume
Gibco BRL 5× Forward buffer 6 ml
T4 polynucleotide kinase 2 ml
γ-32P-ATP 5 ml
DNA oligonucleotide (25mer, 500mM 83mM) 5 ml
ddH2O 12 ml
Total 30 ml

Procedure

  • Incubate the reaction mixture @ 37°C for 15 min up to 1 hr. Invert the column sharply several times to resuspend the settled gel and remove any bubbles.
  • Snap off the tip and place the column in a 2ml microcentrifuge tube.
  • Drain the buffer from a Bio-Spin 6 column.
  • Wash the column with ddH2O.
  • Centrifuge the column for 2 min at 1000×g to remove the remaining liquid.
  • Switch to a clean collection tube.
  • Heat-inactivate the T4 polynucleotidekinase @ 95°C.
  • Pipet 20ml of ddH2O to the labeling sample.
  • The final concentration of the oligo is now 50mM.
  • Take 1ml of pre-column reaction sample and add 2ml of ddH2O.
  • Load the labeling sample onto the Bio-Spin 6 column.
  • Spin the column for 4 min @ 1000× g to remove unincorporated nucleotides.
  • Take 1ml of pro-column reaction sample and add 2ml of ddH2O.
  • Store the samples in –20°C.

Calculating the oligo concentration after labeling.

  • Take out a TLC plate from a designated drawer.
  • Use the tool to etch out 1cm wide sample lanes.
  • Drop ~2ml of pre- and after column *25mer in each lane and let dry.
  • Put in a TLC chamber with 0.3M KH2PO4 pH 3.4 at the bottom.
  • Let the samples migrate to the top of the TLC plate.

Analyze the TLC plate:

  • Dried the plate behind a shield on a sheet of saran wrap.
  • Put in a cassette and exposed for ~15 min.
  • Scanned the plate on the “Storm”.
  • Boxed the first dot. Ctrl+D to copy and moved to the second dot. “Background correct”, “Volume report setup”, “Report” Ratio = (after spin phospho-intensity)/(pre-spin phospho-intensity) Actual oligo concentration = original concentration × ratio

Annealing *25mer to 45mer

  • Combine equal moles of *25mer and 45mer.
  • Put into the 95°C, cool down the heat block on the bench behind a shield.
  • Note: the error rate is reduced when the 25mer is 9:1 cold to hot.

Contributed by Louise Wang.

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