Reagent Preparation Using EpiMark® Bisulfite Conversion Kit (E3318)

Introduction

Add 40 ml of ethanol (96–100%) to Wash Buffer concentrate and store at room temperature (18–25°C). Invert the bottle several times before starting sample clean up procedure. 

Add 27 ml of ethanol (96–100%) to Desulphonation Reaction Buffer concentrate. Store up to 4 weeks at room temperature (18–25°C); for longer than 4 weeks store at 2–8°C. Invert the bottle several times before starting each desulphonation reaction.

Prepare the bisulfite mix by adding 650 μl of nuclease-free water and 250 μl of Solubilization Buffer to solid sodium metabisulfite in the tube provided. Vortex until the bisulfite mix is completely dissolved and clear, usually between 2–5 minutes. If some white particles are still present, heat the solution to 55°–60°C for a few minutes and vortex briefly. Each vial of bisulfite mix is sufficient for 8 reactions. Excess bisulfite mix, that is not used immediately, can be stored frozen for up to 6 months.

Protocol

1. Step I – Bisulfite Conversion Reaction
   Combine the following in a 0.2 ml PCR tube:
   

   Reagent	Volumes per Reaction
   Genomic DNA	10.0 µl (50 ng-2 µg)
   Bisulfite Mix	130.0 µl
   Total Reaction Volume	140.0 µl

2. Step II – Bisulfite Conversion Reaction
   Transfer reaction tube to a thermocycler and begin cycling:
   Denaturation:  95°C  5 minutes
   Incubation:  65°C  30 minutes
   Denaturation:  95°C  5 minutes
   Incubation:  65°C  60 minutes
   Denaturation:  95°C  5 minutes
   Incubation:  65°C  90 minutes
   Hold:  18–20°C  up to 12 hours
   
   
3. Step III – Desulphonation Reaction and Sample Clean Up
   
   1. After completion of the conversion reaction, transfer the individual reactions into 1.5 ml microcentrifuge tubes, add 550 μl of DNA Binding Buffer and mix briefly.
   2. Load the entire sample onto an EpiMark™ spin column with 2 ml collection tube attached. Centrifuge the columns at 15,000 x g for 1 minute and discard the flow through.
   3. Add 500 μl of Wash Buffer, centrifuge the columns at 15,000 x g for 1 minute and discard the flow through.
   4. Add 500 μl of Desulphonation Reaction Buffer to each column and incubate at room temperature (18–20°C) for 15 minutes. Close the lids of the spin columns during incubation.
   5. Centrifuge the columns at 15,000 x g for 1 minute and discard the flow through.
   6. Add 500 μl of Wash Buffer, centrifuge the columns at 15,000 x g for 1 minute and discard the flow through. Repeat wash step.
   7. Centrifuge the columns at 15,000 x g for 1 minute to remove any residual Wash Buffer from the spin columns.
   8. Place the spin columns into sterile 1.5 ml microcentrifuge tubes (not provided). Add 20 μl of Elution Buffer, incubate for 1 minute and centrifuge the columns at 15,000 x g for 1 minute. Add an additional 20 μl of Elution Buffer and spin again for 30 seconds at 15,000 x g.
   The DNA is ready for methylation analysis by PCR. We recommend using 1–6 μ l of DNA for each PCR reaction.