In Vitro DMS Footprinting

 

  1. Mix 3X transcription salts, 0.1-10 ng of DNA and water to a final volume of 100 uL.
  2. Add protein.
  3. Add 6.7 uL 150mM DMS and shake at 37˚C for 5 min.
  4. Add 200 uL cold stop buffer (3M NaOAc, 1M BME, 250 ug/mL yeast tRNA, 20mM EDTA).
  5. Add 600 uL  cold 95% EtOH.
  6. Let sit at -80˚C for 10 min.
  7. Spin at 4˚C for 10 min at max speed.
  8. Wash pellet with 70% EtOH and dry in speedvac on low heat.
  9. Resupend in 100 uL 1M piperdine.
  10. Heat at 90˚C for 30 sec.
  11. Chill on ice and spin through a 1ml Sephadex G50-80 column equilibrated with sterile water.
  12. To 35 uL ~0.5 ng of DNA in water add 1 uL 32P labeled primer (0.3-0.5x106cpm).
  13. Add 1/9 volume (4X) 0.01M NaOH and mix well.
  14. Heat at 80˚C for 2 min and then chill on ice for 5′.
  15. Add 1/9 volume (5X) freshly made 10X TMD buffer (0.5M Tris-HCl pH 7.2, 0.1M MgSO4, 2mM DTT).
  16. Incubate at Tm of primer (45-50˚C) for 3′ and then chill on ice.
  17. Add 1/9 volume (5X) of mix of dNTPs with 5mM of each dNTP.  Tap tubes and place on ice.
  18. Add 1 uL of diluted Klenow fragment and tap tubes for 10 seconds.
  19. Incubate at 50˚C for exactly 10 min and then add 1/3 volume Quench(4M NaOAc, 20mM EDTA).
  20. Mix thoroughly and precipitate with 2.5-3 volumes cold 95% EtOH.
  21. Let sit at -80˚C for 20 min or O/N at -20˚C.
  22. Spin at max speed at 4˚C for 10 min.
  23. Wash pellet gently with 70% EtOH by rolling tube.
  24. Spin at max speed at 4˚C for 2 min.
  25. Aspirate liquid and dry pellet in speedvac on low heat.
  26. Run 6% acrylimide gel.