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Finkelstein Lab Wiki

Radioactive End Labeling

The Purpose: Use Gamma 32P-ATP to radioactively end-label Lambda DNA HindIII Digest Ladder for use in Radioactive Gels.

Contents

Section titled “Contents”
  1. End-labeling reaction
  2. Alkaline ladder
  3. Alkaline gel loading dye (6X)
  4. References

1. End-labeling reaction

Section titled “1. End-labeling reaction”

1.1 Reaction mix

Section titled “1.1 Reaction mix”

Make a 40 ul reaction:

  • 29 ul diH2O
  • 4 ul 10x T4 PNK Buffer (*contains MgCL2)
  • 4 ul NEB Lambda DNA HindIII Digest Ladder [500ng/ul]
  • 2 ul T4 PNK Enzyme
  • 1 ul Gamma 32P-ATP
  • 40 ul Total Volume

1.2 Incubation and cleanup

Section titled “1.2 Incubation and cleanup”
  1. Incubate reaction 1 hour at 37°C.
  2. Heat inactivate T4 enzyme at 65°C for 20 minutes. Cool.
  3. Apply reaction (50 ul max volume) to Monarch PCR Clean Up Kit for larger than 50 bp to remove unincorporated Gamma 32P-ATPs.
    1. Discard radioactive column in radioactive dry waste.
  4. Store radioactive end-labeled DNA Lambda HindIII ladder in radioactive -20°C freezer.

2. Alkaline ladder

Section titled “2. Alkaline ladder”
  • 6ul Lambda HindIII end-labeled ladder
  • 9 ul diH2O
  • 3 ul 6X Alkaline Loading Dye
  • 18 ul Total Volume

Load 2-6 ul on Alkaline Agarose gel.

3. Alkaline gel loading dye (6X)

Section titled “3. Alkaline gel loading dye (6X)”
  • 45 ul 10M NaOH
  • 18 ul 0.5M EDTA
  • 0.33g Ficoll 400
  • 0.003g Bromocresol Green Dye
  • 0.004g Xylene Cyanol Dye
  • QS to 1.5 mls with diH2O

4. References

Section titled “4. References”