From tissue to sequence: These are the general protocols we use for DNA extraction, PCR and sequencing
1. Extraction: We use the standard phenol extraction protocol
2. Quantitation of genomic DNA: we use the spectrophotometer or the fluorometer
3. PCR amplification (see general protocol). In general, 50 uL reactions seem to give better recovery after clean-up, especially with large fragments.
4. If you get a single strong band, proceed to DNA cleanup. In most cases, Gibco Concert PCR prep kit works well, but if you have a large fragment (e.g. 1.5 kb or more) or the total quantity of DNA in the PCR product is large, ethanol precipitation may work better.
5. Quantitate purified PCR products in the fluorometer
6. Perform cycle sequencing reaction (BigDye Terminator). Use this chart to determine the correct quantity of purified template DNA needed depending on fragment size (this chart is also posted at various places around the lab).
7. Purify BigDye reaction products using the ethanol precipitation protocol. (Note that the EtOH precipitation protocols for PCR and BigDye products are slightly different)
8. Prepare the samples for the ABI-310 as follows:
