EpiTect Bisulfite Handbook

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19. Place the spin column into a clean 1.5 ml microcentrifuge tube (not provided). Add 20 µl Buffer EB to the center of the membrane. Elute the purified DNA by centrifugation for 1 min at approximately 15,000 x g (12,000 rpm.) Note: To increase the yield of DNA in the eluate, transfer the spin column to a new 1.5 ml microcentrifuge tube, add an additional 20 µl Buffer EB to the center of the membrane, and centrifuge for 1 min at maximum speed. Combine both eluates. Note: If the purified DNA is to be stored for up to 24 hours, we recommend storage at 2–8°C. For storage longer than 24 hours, we recommend storage at –20°C. At –20°C, DNA converted and purified using the EpiTect Bisulfite Kit can be stored for longer than 12 weeks* without decrease of quality or conversion. * Investigations into longer storage of converted DNA are ongoing. Contact QIAGEN for more information. 28 EpiTect Bisulfite Handbook 04/2006
Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or molecular biology applications (see back page for contact information). Comments and suggestions Little or no DNA recovery in purification step a) Carrier RNA not added to Buffer BL b) Buffer BW or Buffer BD prepared incorrectly c) Buffer BW or BD prepared with 70% ethanol d) Buffer BW and Buffer BD used in the wrong order e) Sample not completely passed through the membrane f) Buffer BL contains precipitates Low conversion rate a) Bisulfite reaction components not added in the correct order b) Incorrect thermal cycling conditions used Prepare carrier RNA and add to Buffer BL, as described in “Things to do before starting”, pages 15, 20, and 24. Check that Buffer BW and BD concentrates were diluted with the correct volumes of ethanol (96– 100%). Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. Check that Buffer BW and BD concentrates were diluted with 96–100% ethanol. Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. Ensure that Buffer BW and Buffer BD are used in the correct order in the protocol. Centrifuge for 1 min at full speed or until the entire sample has passed through the membrane. Check Buffer BL for precipitate. Dissolve by heating (maximum 70°C) with gentle agitation. Ensure that DNA, Bisulfite Mix, and DNA Protect Buffer are added in the order indicated in Table 2, page 17, Table 4, page 21, or Table 6, page 25. Use the thermal cycling conditions given in Table 3, page 17, Table 5, page 22, or Table 7, page 26. EpiTect Bisulfite Handbook 04/2006 29
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 and 14: Important Notes Yield and size of D
Page 15 and 16: Protocol: Sodium Bisulfite Conversi
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: 7. Add 560 µl freshly prepared Buf
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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