Genomic DNA Preparation Troubleshooting
Some of the more common problems that can occur during genomic DNA preparation are described in Table 4.4. Refer to the instructions provided with your genomic DNA preparation product for more detailed troubleshooting information.
| Problem | Possible cause | Solution |
|---|---|---|
| Quality of genomic DNA is poor (genomic DNA is not high molecular weight or looks degraded) | Starting sample was not stored properly. Starting sample was fixed with formalin or another fixative (e.g., FFPE tissue). Purified genomic DNA was not stored properly.
| Nucleases may have degraded genomic DNA. Follow recommendations for storage and handling of your sample type. Genomic DNA may be fragmented in the sample. It is not possible to obtain high-molecular-weight DNA from this sample type. Follow recommended storage conditions to minimize degradation by nucleases. Do not store DNA in water for extended periods. |
| DNA yield is low | If the yield is low and purity/quality is good: Starting sample size was insufficient. If yield and purity/quality are low: Starting sample was not stored properly. Cells were not lysed thoroughly. | Limited samples such as buccal swabs, FTA/Guthrie card blood, etc. may require amplification of genomic DNA using Phi29 DNA polymerase. Nucleases may have degraded genomic DNA. Follow recommendations for storage and handling of your sample type. Follow homogenization/lysis recommendations for your sample type. |
| A260/A280 of genomic DNA sample is < 1.7 or > 1.9 | A260/A280 < 1.7: Proteins or chaotropes may be present. A260/A280 > 1.9: RNA may be present. | Process starting sample according to recommended instructions to ensure thorough removal of proteins. Do NOT overload the purification system. To remove protein contaminants, treat the sample with a protease or perform a phenol extraction + ethanol precipitation. Ensure wash steps are followed closely to eliminate carryover of contaminants into your final sample. Follow recommendations for processing different sample types. Some sample types contain high levels of RNA. RNA may not interfere with some applications. |
| DNA does not work well in downstream application | Genomic DNA quality is poor. If DNA quality is good: Contaminants (lipids, salts, etc.) may be present in the sample. For silica purification using ethanolic wash buffer: Residual ethanol remains in the sample. | See troubleshooting tips above for poor-quality genomic DNA. Do not overload the purification system. This may result in poor yield and/or decreased purity of genomic DNA. If organic solvents such as phenol were used, residual solvent may be present in the sample. Precipitate the DNA with salt and alcohol. (Appendix 4.) Be sure to thoroughly air dry the sample prior to elution.
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Materials
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