Multi-Nuclease Protection Assay
Dominik Haudenschild May 11,1998
Kinase-Max™ Oligo Labeling

• __5_µl Water (Nuclease-free)
  
• __1_µl 10pmol Oligo (at 10 pmol/µl = 10µM)
  
• __2_µl 5x forward buffer
  
• __1_µl [g³²P] ATP at 150 mCi/ml (NEG-035C)
  
• __1_µl T4 PolyNucleotideKinase (10U/ml)
  

• Make Digestion Mixture = 1x Digestion Buffer, 1:200 dilution of Nuclease Mixture
  
• Add 200µl of Digeston Mixture to each reaction
  - negative control gets 200µl Digestion Buffer without Nuclease
  
• Vortex & spin briefly, incubate at room temperature for 45 minutes
  - at 20 minutes, Vortex & Centrifuge briefly
  
• Add to 1.5ml tube with 40µl Inactivation Buffer and 600µl Ethanol, vortex & spin
  
• Precipitate at -20°C for 15 minutes, then centrifuge ≥15 minutes at 4°C top speed
  
• Aspirate supernatant (pellet is blue)
  
• Wash with 70% ethanol, aspirate, and air-dry pellet for 5-10 minutes
  
• Dissolve in 15µl Gel Loading Buffer by heating to 95°C and vortexing
  
• Load onto 15% acrylamide TBE-Urea gels
  Bromophenol Blue runs at about 15 bases, Xylene Cyanol at about 60 bases
  
• Soak gel in 30% Methanol, 5% Glycerol 30-45 minutes to prevent gel cracking
  
• Dry under vacuum at 60°C for at least two hours
  
• Expose to phosphorimaging screen

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