| Successful sequencing reactions are very much dependent on the DNA template. Both
quality and concentration are critical to ensure long reads. User submitted samples for
DNA sequencing must be of high quality and submitted at a concentration of 250 ng/ul. The most important factor in DNA sequencing is template quality. Because RNA, phenol carry-over and other contaminates affect sequence quality, precautions need to be taken when preparing DNA templates for sequencing. We have included two mini-prep protocols that we have found useful to achieve the necessary quality required for sequencing. Our preferred method for doing plasmid mini-preps is outlined in the alkaline lysis procedure. A more involved procedure developed at UC Davis is included as an alternative. In both cases, steps are included to deal with the two most common inhibitors of sequencing reactions, contaminating RNA and phenol carry-over. Several methods for estimating DNA concentration can be used for submitted samples. These include: densitometry (spot density from ethidium gel), visual estimation (from ethidium gel), UV quantification, and fluorimetry. While fluorimetry is probably the most reliable, not all users have access to this type of equipment and so we recommend densitometry (against known DNA mass ladder standards). We do not recommend UV estimation because of the effect of UV-absorbing contaminants have on over estimating DNA concentration. If users visually estimate DNA concentration from an ethidium gel, a photo (which includes an appropriate moleculer standard) must be included when submitting samples. For users who do their own reactions, Licor's SequiTherm Cycle Sequencing protocol is included and should be noted for the subtle but significant change in the denaturing temperature from 95 C to 92 C used in the thermocycle profile. |
[Alkaline lysis miniprep | Sephacryl Sequencing Miniprep | SequiTherm Cycle Sequencing
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