1-Remove template plates from freezer and let thaw (approximately 10 min).
2-Place each template plate in a 9600 PCR reaction tube base and centrifuge at 3,000 x g for 2 min (program#40 on the Jouan centrifuges) to spin down any droplets on the top or sides of tubes.
3-Set up empty PCR reaction tubes in a 9600 tray/retainer/base set.
4-Add 5 µL of template to PCR reaction tubes.
Note: It is easiest to remove template by poking through the foil cover on the template plate with the tip of a Pipetman. Make sure at least 1 µl of template remains in the tube after removing the 5 µl for the sequencing reaction.
5-Remove sequencing reagents from the freezer and let thaw. Once thawed, place immediately on ice.
·Terminator mix (contained in the Applied Biosystems PRISM READY REACTION Dye Deoxy Terminator Cycle Sequencing kit)
M13 (-21) universal primer (contained in the kit), or:
a unique sequencing primer, at a concentration of 0.8 pM/µl
6-To the 5 µl template add:
Note: To save time and pipetting steps, you can prepare a sequencing "cocktail" containing terminator mix and H2O (and primer, if many of the reactions use the same primer), then add the appropriate amount of cocktail to each reaction tube.
7-Immediately place tubes in the 9600 thermal cycler and start program 7.
Program 7 cycle parameters:
96 °C, 15 sec
50 °C, 1 sec
60 °C, 4 min
repeat for 25 cycles, then:
4 °C hold forever
(total run time for program 7 is approximately 2.5 hr)
Purification of Sequencing Reactions
The excess dye-labeled dideoxy dNTP's must be separated from the sequencing reaction products. This is accomplished by running the reactions through mini Sephadex G-50 columns, in a 96-well format.
1-Rehydrate Sephadex G-50 Fine (Pharmacia, product # 17-0573-02) by adding approximately 15 mL dH2O per gram of Sephadex and autoclaving, or heating at 90° C for 1 hour. Alternatively, the Sephadex can be rehydrated overnight at room temperature.
Note: To discourage bacterial and fungal growth, rehydrated Sephadex should be stored at 4° C and warmed to room temperature before use. Always check for contamination before using. Sodium azide may be added as a preservative (add 1 /1000 volume of a 5% sodium azide stock in water); however, sodium azide is extremely toxic and must be handled with great care and disposed of properly.
2-Remove excess H2O from the Sephadex beads with a pipette (washing to re move fine particles is not required).
3-Add approximately 350 µl of the Sephadex slurry to the wells of a Silent Monitor 96-well 0.45 µm loprodyne filter plate (Pall Europe Ltd., Portsmouth, England, product # SMO45LP). If only processing one plate, prepare a centrifuge balance plate by adding water to the wells of another Silent Monitor plate.
4-Place each plate on top of two paper towels folded in half (to soak up the water that spins out) and centrifuge at 1500 x g for 2 min (program 41 on the Jouan centrifuges).
5-Repeat steps 3 and 4 until wells are full and Sephadex is flush with the top of the plate. If wells become too full, scrape off the excess Sephadex. Keep the same plate orientation for each spin, and be sure to change paper towels as neces sary.
6-Set up empty 9600 PCR reaction tubes in a tray/retainer/base set.
7-Place the Silent Monitor plate on top of the PCR reaction tubes.
8-Pipet each of the 20 µl dye terminator reactions onto the Sephadex resin. Be very careful to load the samples slowly, and pipet directly into the center of each column.
9-Centrifuge at 750 x g for 2 min (program 42 on the Jouan centrifuges). Spin plates in the same orientation as before.
10-Remove and discard the Silent Monitor plate. The Sephadex resin should be pink and the eluant colorless.
11-Dry samples in a vacuum dessicator and resuspend in gel loading buffer.