Fan Lab Bisulfite Treatment Protocol

By Shaun Fouse

Make sure to wear gloves, eye goggles and a lab coat during the procedure!

Day 1

  1. Digest at least 5ug of DNA overnight with your favorite enzyme.  Make sure that the enzyme does not cut within the region you plan to analyze.  We have found that Bgl II works well for most of our analyses.  I usually set up a 50 uL reaction.
  2. Run a quick check gel of your digest.  You need this to decide how much of your digested product to treat.

Day 2

  1. Heat a total of 20uL of DNA and H2O (depending on the DNA concentration) at 97º for 5 min.  If you do this in PCR tubes, you can do this step in the PCR machine.
  2. While this is going on, make up a 10 mL solution of 6.3M NaOH (2.52g in 10mL of H2O).  This will be used tomorrow as well.
  3. After the DNA has been heated, chill on ice for 1 min and then add 1uL of 6.3M NaOH Ð mix well.
  4. If not already in PCR tubes, transfer to PCR tubes now
  5. Place DNA in PCR machine and start BS program (on the downstairs PCR machine MJ1 the program is BS2) ­ let it heat up to 39º and then pause program.  Set timer for 30 min and start it.
  6. Start making up Sodium Bisulfite Solution.  For sodium bisulfite, dissolve 4.05 g of the Sodium Bisulfite in 8mL of dH2O.  Wrap in foil as this solution is light sensitive and then place at 55º to help it dissolve.  To make the Hydroquinone, dissolve 0.11g of hydroquinone in 5mL of dH2O.  Wrap this up in foil as well and heat at 55¡ to help dissolve. 
  7. When both solutions are dissolved, add 330uL of the hydroquinone solution to the Sodium Bisulfite.  Also add 333uL of the 6.3M NaOH.
  8. After the DNA has incubated at 39º for 30 min, add 208 uL of the sodium bisulfite solution to each tube Ð make sure to mix well.
  9. Un-pause the PCR machine and let the PCR program run for 15-16 hours.  We usually do this overnight.  After 16 hours, the bisulfite starts to degrade the DNA.

Day 3

  1. Desalt the DNA using the Promega Wizard DNA Cleanup kit. 
  2. Set up and label the tubes and columns before you start
  3. Shake up the resin and pipet 1mL to a new eppie.  Add your bisulfite DNA to the tube and mix by inverting
  4. Add this to the column and pull through with the vacumn.
  5. Wash with 2mL of 80% isopropanol.  Start with 1mL in the column and then add another mL when the isoproanol is almost out.  Let the vacumn run for 30 sec after it has pulled all the liquid through
  6. Transfer column to new eppie and spin at max for 2 min to remove remaining isopropanol
  7. Transfer to a new eppie and add 50uL of TE that has been prewarmed.  Let it stand for 1 min and then spin at max for 30 sec.
  8. After collecting DNA, add 2.5uL of 6.3M NaOH.  Mix well and incubate at 37º for 15 min
  9. Spin down DNA and then add 22.5 ul of 10M NH4OAC, 0.5 uL of glycogen and 225 uL of 100% EtOH to ppt DNA.  Vortex and spin at max for 15 min.
  10. Carefully remove the supernatant and wash with 1 vol of 70% EtOH.  Vortex gently to wash pellet.  Spin at max for 1 min.
  11. Carefully remove supernatant and let pellet briefly dry. 
  12. Resuspend DNA in 30-50 uL of TE