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œœœœœ@DMS-treating DNA@œœœœœ



@DMS treatment under controlled conditions, followed by piperidine cleavage, yeilds a ladder of fragments indicating the positions of all G residues in the DNA sequence. These ladders are useful MW standards for S1 experiments, footprinting, etc.

  1. Reagents

    1. DMS buffer
      50mM Na Cacodylate,pH8
      1mM EDTA

    2. Sequencing dye
      80% formamide
      10mM NaOH
      1mM EDTA
      0.1% xylene cyanol FF
      0.1% bromophenol blue

    3. DMS STOP
      1.5M NaOAc
      1M 2-ME

    4. Carrier DNA
      @1mg/ml sonicated or sheared salmon sperm DNA

  2. Methods

    1. Dry the desired amount of singley end-labeled DNA in an eppendorf tube (you need 5000-20,000 CPM per lane on a gel).
    2. Resuspend the DNA in 200ul ice-cold DMS buffer. Working on ice, add 3ul carrier DNA, vortex, and add 1ul DMS. Vortex again, and incubate 5 min on ice.
    3. Add 50ul DMS STOP and 750ul EtOH, mix, and freeze for 5 min in a dry-ice:EtOH bath. Spin for 5 min and discard the supernatant.
    4. Dissolve the pellet in 250ul 0.3M NaOAc, then add 750 EtOH and freeze & spin as before. Add 1ml ice-cold 70% EtOH to the pellet, and freeze & spin again. Discard the supernarant and dry the pellet in a roto-vac. Add 20ul ddH2O to the dry pellet and dry again.
    5. Add 100ul 1:10 piperidine (freshly diluted in ddH2O) and incubate for 30min at 90C. Quick-freeze the solution in a dry-ice:EtOH bath, then dry the sample in a roto-vac (with the heat ON to speed the drying).
    6. Add 10ul ddH2O to the dry pellet, and redry. Repeat this step.
    7. Count the pellet and resuspend the DNA in enough sequencing dye for 3-5ul per lane. Remember to heat the sample for 5min at 90C, then cool on ice before loading the gel.


Meth Enzymology 65, Maxam & Gilbert

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