Sequencing reactions

For sequencing, we use ABI's Big Dye v3.1 sequencing kit. The sequencing mix is 2.5x. It can be diluted with sequencing buffer (stored in 4C fridge). We make up the sequencing reactions in the skirted plates that go on the ABI machine (either ABI plates or USA Scientific TempPlate III #1402-9700). Typical dilutions for a 20 ul reaction would go like this: Reagent Seq buffer (5x) Primer (3.2 uM) DNA Template Water Full rxn 1/2 1/4 1/8 4 2 1 x 2 3 1 x 1 3.5 1 x 1 x 11-x 1/16 0.5 3.75 1 x 20 1/16 0.25 1.875 1 x

Term Rdy Mix (2.5x) 8 ul

13-x 14-x 14.5-x 14.75-x

Total volume 20 ul 20 20 20 For a 10 ul reaction, it would be: Reagent Full rxn 1/2 1/4 1/8 Term Rdy Mix (2.5x) 4 ul Seq buffer (5x) Primer (3.2 uM) DNA Template Water 1 x 5-x 2 1 1 x 1 1.5 1 x 0.5 1.75 1 x

6-x 6.5-x 6.75-x 6.875-x

Total volume 10 ul 10 10 10 10 Run cycle sequencing protocol on PCR machine: 94 15s / 50 C 4s / 60 C 2 min 25 x. Sequencing reaction clean up: You can add the following to the PCR reactions in the skirted PCR plates. However, if you ran the sequencing reaction in some other tubes, you can now do the clean up in the skirted plates so it is all ready to put on the sequencer. We use the sodium acetate / EDTA / Ethanol precipitation clean up. To each well add: Reagent 10 ul rxn 20 ul rxn 125 mM EDTA 1 ul 3 M NaOac 1 ul 2 ul 2 ul

100% EtOH 25 ul 50 ul These can be premixed and then added together. Next spin to precipitate and wash: 1. 2. 3. 4. 5. 6. 7. Incubate at RT for 15 min. (Can be left O/N or several days at -20C at this step). Spin in Allegra centrifuge 1650g, 4 C for 45 min. Invert and spin onto paper towel 185g 2 minutes Wash with 70 ul of 70% EtOH. Spin 1650G, 4C for 15 min. Invert and spin onto paper towel, 185g 2 minutes. Can speed vac to dry if need be. Add Hi-Di and seal with septa seal plate. Now its ready to load onto sequencer.