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
- γ–32P_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)
