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 |
Full rxn |
1/2 |
1/4 |
1/8 |
1/16 |
| Term Rdy Mix (2.5x) |
8 ul |
4 |
2 |
1 |
0.5 |
| Seq buffer (5x) |
- |
2 |
3 |
3.5 |
3.75 |
| Primer (3.2 uM) |
1 |
1 |
1 |
1 |
1 |
| DNA Template |
x |
x |
x |
x |
x |
| Water |
11-x |
13-x |
14-x |
14.5-x |
14.75-x |
| Total volume |
20 ul |
20 |
20 |
20 |
20 |
For a 10 ul reaction, it would be:
| Reagent |
Full rxn |
1/2 |
1/4 |
1/8 |
1/16 |
| Term Rdy Mix (2.5x) |
4 ul |
2 |
1 |
0.5 |
0.25 |
| Seq buffer (5x) |
- |
1 |
1.5 |
1.75 |
1.875 |
| Primer (3.2 uM) |
1 |
1 |
1 |
1 |
1 |
| DNA Template |
x |
x |
x |
x |
x |
| Water |
5-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: We use the sodium acetate / EDTA / Ethanol precipitation clean up. For each well add:
| Reagent | 10 ul rxn | 20 ul rxn |
| 125 mM EDTA | 1 ul | 2 ul |
| 3 M NaOac | 1 ul | 2 ul |
| 100% EtOH | 25 ul | 50 ul |
These can be premixed and then added together. Next spin to precipitate and wash:
- 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.