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== Bisulfite conversion == * Performed bisulfite conversion using EZ DNA Methylation-Gold™ Kit and did the same procedure following manufacturer's instruction and elute with 42ul Elution buffer. This kit is the same kit as LifeTech MethylCode™ Bisulfite Conversion Kit. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :- Prepare 3 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer :- Mix well and spin down the tube :- Put on thermomixer at 37C for 15min, 1,000rpm :- Solution should be clear :- Spin down the tube :- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul :- Mix by pipetting 10X with multi-channel pipette :- Spin down the tube :- Incubate following below program ::- 98°C for 10 minutes (DNA denaturation) ::- 64°C for 2.5 hours (Bisulfite conversion) ::- 4°C storage for up to 20 hours or continue to desulfonation :- Mix 600ul of Binding Buffer with 1ul of 10ng/ul tRNA. For many reaction, make multiple mix. :- Add 601ul Binding Buffer and tRNA mix to each column :- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column :- Mix by pipetting 5x :- Spin down column at 14,000 rpm for 30s :- <u>Discard spnt</u> :- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube) :- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> :- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube) :- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> :- Add 200ul Wash Buffer :- Spin down column at 14,000 rpm for 30s :- <u>Discard spnt</u> :- Add 200ul Wash Buffer :- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u> :- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube :- Add 32ul of Elution Buffer (warm Elution Buffer at 60C for 10min) :- Wait for 2min :- Spin down column at 12,000 rpm for 1min :- Continue to amplification (During elution step, PCR mix should be prepared)
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