EtOH / EDTA Precipitation - Cycle Sequencing
1. Reaction volumes are assumed to be 10 μl.
2. 2.5 μl 125mM EDTA.
3. 27.5 μl 100% EtOH – high quality such as from Sigma.
4. Mix gently by inverting 3 or 4 times or vortex at speed 7 (Fisher basic vortexer).
5. Wait for 15 minutes.
6. Centrifuge for 30 minutes at 2000+ RCF at 4ºC.
7. Immediately after centrifugation, uncover plates, invert it onto kimwipe to drain wells from alcohol and centrifuge for 1 minute at 140 RCF or lower.
8. Add 30 μl 70% EtOH.
9. Centrifuge for 15 minutes at 1650 RCF (at 4ºC or room temperature).
10. Immediately after centrifugation, uncover plates invert it onto kimwipe to drain wells from alcohol and centrifuge for 1 minute at 140 RCF.
11. Leave uncovered on bench for an hour to dry or put uncovered in a 37ºC incubator for ~15 minutes.
12. Close tubes, store in cool and dry place until being resuspended and being resolved on a sequencer.
13. Once ready, resuspend samples in 10 ul Hi-Di Formamide, gentry vortex, denature at 94ºC (heat denature is optional since Formamide is denaturing), and load onto the sequencer.