DNA oligonucleotide 5’-labeling with 32P

DNA oligonucleotide 5’-labeling with 32P

 

1- Prepare 5X Kinasing Buffer:

  • 1 M Tris-HCl pH 7.5: 12.5 uL
  • 1 M MgCl2: 2.5 uL
  • 1M DTT: 1.25 uL
  • 10 mM Spermidine: 2.5 uL
  • 200 mM EDTA: 2.5 uL
  • H2O: 30.75 uL

2- Kinase mix:

  • 5X Kinasing Buffer: 4 uL
  • γ32P_ATP: x  uL        (30 to 50 pmoles)
  • DNA: y uL        (3 to 5 pmoles)
  • 10U/uL T4 Polynucleotide Kinase: 1 uL
  • H2O: 15-x-y   uL

3- Incubate 30 min @ 37 C then add 2 uL of 200 mM EDTA.

4- Spin one Biorad MicroBiospin P30, 2 min, 1020 g without cap.

5- Add 500 uL EtOH 20 %. Spin 1 min @ 1020g. Repeat.

6- Wash 3X with H2O, and then 3X with TE.

7- Apply the kinasing mix and spin 4 min @ 1020 g.

8- Complete the volumes recovered to 100 uL with TE.

9- Count 1 uL using the liquid scintillation counter and report the cpm/uL on the tube and store @ -20 C.

NB:

[ATP total] = [ATP hot] + [ATP cold] = (16.5 x D + 8.5) μM

(Where D is the Decay factor between Today and the Calibration Date)

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