EpiTect Bisulfite Handbook

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c) Poor DNA quality (i.e., protein contamination) d) Amount of DNA used outside recommended range e) Bisulfite Mix stored incorrectly f) DNA Protect Buffer not added Comments and suggestions Check that the A260/A280 ratio of the sample DNA is between 1.7 and 1.9. Ensure that sample DNA is purified using an appropriate kit (see ordering information, pages 32–34 for suitable QIAGEN kits for DNA purification). Increase or decrease the amount of starting DNA material to stay within the range of 1 ng to 2 µg DNA for standard applications, 1 ng to 500 ng for low concentrations of DNA in solutions (page 20), or 1 ng to 2 µg for DNA from FFPE tissues (page 24). Dissolved Bisulfite Mix can be stored at –20°C for 4 weeks. Upon addition of DNA Protect Buffer, the DNA– Bisulfite Mix solution should turn from green to blue indicating sufficient mixing and the correct pH for DNA binding to the EpiTect spin column. If this color change does not occur, repeat the reaction ensuring that DNA Protect Buffer has been added. Poor results in downstream methylation-specific PCR a) Little or no PCR product even in control reaction If performing hot-start PCR, confirm that the initial enzyme activation step was performed. Ensure that all PCR components were added and that suitable cycling conditions were used. 30 EpiTect Bisulfite Handbook 04/2006
) Failure of conversion reaction Unexpected findings in buffers a) Color of DNA Protect Buffer changes from light green to olive during storage, b) Precipitates in Buffer BD Comments and suggestions The starting DNA was not sufficiently pure. Ensure that only high-quality DNA is used for the conversion reaction. See ordering information, pages 32–34 for suitable QIAGEN kits for DNA purification. Ensure that all steps of the modification and cleanup protocol were followed. Sample DNA was degraded before modification reaction. Ensure that sample DNA is handled and stored correctly. PCR primers were not appropriate or incorrectly designed. Check primer design. Amount of template DNA used in PCR reaction was insufficient. Increase amount of template DNA. DNA Protect Buffer is stable at room temperature (15–25°C) for one year, and a change in color within this time has no influence on performance. There may be slight clouding and/or insoluble precipitates in Buffer BD during storage. Buffer BD is stable at room temperature (15– 25°C) for one year, and a precipitate within this time has no influence on performance. Precipitates should not be transferred onto the membrane. EpiTect Bisulfite Handbook 04/2006 31
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 and 28: 7. Add 560 µl freshly prepared Buf
Page 29: Troubleshooting Guide This troubles
Page 33 and 34: Product Contents Cat. no. MagAttrac
Page 35 and 36: QIAGEN Distributors and Importers P

EpiTect Bisulfite Handbook

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