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Bisulfite Treatment
Fan Lab Bisulfite
Treatment
By Shaun Fouse
Reminder ­ Wear Gloves, lab coat and eye protection!!!
Day 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.
- Run a
quick check gel of your digest.
You need this to decide how much of your digested product to treat.
Day 2
- 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.
- 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.
- After
the DNA has been heated, chill on ice for 1 min and then add 1uL of 6.3M
NaOH Ð mix well.
- If not
already in PCR tubes, transfer to PCR tubes now
- 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.
- 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.
- When
both solutions are dissolved, add 330uL of the hydroquinone solution to
the Sodium Bisulfite. Also
add 333uL of the 6.3M NaOH.
- 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.
- 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
- Desalt
the DNA using the Promega Wizard DNA Cleanup kit.
- Set up
and label the tubes and columns before you start
- Shake
up the resin and pipet 1mL to a new eppie. Add your bisulfite DNA to the tube and mix by inverting
- Add
this to the column and pull through with the vacumn.
- 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
- Transfer
column to new eppie and spin at max for 2 min to remove remaining
isopropanol
- 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.
- After
collecting DNA, add 2.5uL of 6.3M NaOH. Mix well and incubate at 37º for 15 min
- 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.
- Carefully
remove the supernatant and wash with 1 vol of 70% EtOH. Vortex gently to wash pellet. Spin at max for 1 min.
- Carefully
remove supernatant and let pellet briefly dry.
- Resuspend
DNA in 30-50 uL of TE
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