DNA purification by CsCl ultra-centrifugation

This protocol is for 1 ml polycarbonate ultra-centrifuge tubes. I obtained this basic protocol from Lee Kerkhof, who uses a similar setup for Stabile Isotope Probing (SIP).

• Re-suspend precipitated DNA in 50 ul DI (DEPC treated for RNA samples)
• Make a CsCl master mix (N+1, where N=# of samples)

   DI     460 ul  x N
   CsCl   0.54g  x N
   Ethidium Bromide solution   0.5 ul  x N

• Make sure the CsCl is dissolved completely
• Add 610 ul of CsCl mix to each tube
• Add DNA (50 ul) to respective tubes
• Mix well by using a small piece of parafilm and inverting several times
• place styrofoam holder on balance and tar
• Balance paired tubes to 0.001g by removing liquid from the heavier tube using a 10 ul pipette.
• Load tubes into rotor (Remember to put balanced pairs in opposite slots !!!)
• Spin samples at 80,000rpm in benchtop ultra-centrifuge at 20degC for 16h
• Retrieve tubes and place on transilluminator in holder
• use 100 ul pipette to recover the ethiddium stained DNA band