EpiTect Bisulfite Handbook

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Processing EpiTect spin columns in a microcentrifuge � Always close EpiTect spin columns before placing them in the microcentrifuge. � For efficient parallel processing of multiple samples, we recommend filling a rack with the collection tubes into which EpiTect spin columns can be transferred after centrifugation. Collection tubes can be used several times. 14 EpiTect Bisulfite Handbook 04/2006
Protocol: Sodium Bisulfite Conversion of Unmethylated Cytosines in DNA Using this protocol, 2 µg – 1 ng DNA in a volume of up to 20 µl can be processed. Important points before starting � Each aliquot of Bisulfite Mix is sufficient for 8 conversion reactions. If converting fewer than 8 DNA samples, reconstituted Bisulfite Mix can be stored at –20°C for up to 4 weeks without any loss of performance. � The DNA Protect Buffer should turn from green to blue after addition to DNA/Bisulfite Mix (step 2), indicating sufficient mixing and correct pH for the bisulfite conversion reaction. � Perform all centrifugation steps at room temperature (15–25°C). Things to do before starting � Add 30 ml ethanol (96–100%) to Buffer BW and store at room temperature (15–25°C). Invert the bottle several times before starting the procedure. � Add 27 ml ethanol (96–100%) to Buffer BD and store at room temperature. Invert the bottle several times before starting the procedure and close the bottle immediately after use. White precipitates may form after addition of ethanol. These precipitates will not affect the performance of Buffer BD. However, avoid transferring precipitates to the EpiTect spin column. � Add 310 µl RNase-free water to the lyophilized carrier RNA (310 µg) to obtain a 1 µg/µl solution. Dissolve the carrier RNA thoroughly by vortexing. When processing 48 samples at once, add the complete volume of dissolved carrier RNA to the bottle of Buffer BL, and check the box on the bottle lid label. If processing fewer samples, split the dissolved carrier RNA into conveniently sized aliquots (e.g., 50 µl) and store at –20°C. Aliquots can be stored for up to 1 year. If
Page 1 and 2: EpiTect ® Bisulfite Handbook For c
Page 3 and 4: Contents Kit Contents 4 Shipping an
Page 5 and 6: Unused portions of carrier RNA diss
Page 7 and 8: 24-hour emergency information Emerg
Page 9 and 10: DNA Protect Buffer that contains a
Page 11 and 12: DNA Protect Buffer DNA Protect Buff
Page 13: Important Notes Yield and size of D
Page 17 and 18: Table 2. Bisulfite Reaction Compone
Page 19 and 20: 15. Place the spin column into a ne
Page 21 and 22: � Equilibrate samples to room tem
Page 23 and 24: 9. Centrifuge the column at maximum
Page 25 and 26: � Add dissolved carrier RNA to Bu
Page 27 and 28: 7. Add 560 µl freshly prepared Buf
Page 29 and 30: Troubleshooting Guide This troubles
Page 31 and 32: ) Failure of conversion reaction Un
Page 33 and 34: Product Contents Cat. no. MagAttrac
Page 35 and 36: QIAGEN Distributors and Importers P

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