Sequence with Big Dye Kit (PE-biosystem) and ABI PRISM 310 Genetic Analyzer

- plasmid miniprep of 3-5 ml LB-culture
- QIAGEN kit, elute with water (pH above 7; or with 10 mM Tris/no EDTA!)
- ethanol precipitation may be ommitted:
- 0,1 vol. 3 M NaAcetate pH 5
- add 2,2 Vol. ethanol
- 15 min, 13 krpm
- add 70% ethanol to pellet; 3 min 13 krpm
- dry a bit in the air at RT (or speedvac or 90°/1 min); add 50 µl water

PCR amplification followed by purification via QIAquick PCR Purification Kit (QIAGEN), can also be used instead of the procedure illustrate above (usefull when working with some bacterial strains as E.Coli TG1)

- mix the following 5 µl reaction assay components on ice in thin sequence tubes
- 1 µl 0,8 pmol/µl primer
- 2 µl template (plasmid miniprep; around 120 ng (or more))
- 2 µl Big Dye Taq Mix;
- dont use oil; preheat lid 110° on;

• 96°  1 min
• 96°  10 sec
• 50°  5 sec
• 60°  4 min; back to step2; 25 cycles
• 4°  24 h
• temperature ramp: < 1°/sec

- add 15 µl water; add 2 µl 3 M NaAcetate pH 5; add 50 µl ethanol
- 13 krpm 15-30 min (evtl. 4°); remove supernatant very carefully; pellet is invisible
- rinse with 70% ethanol; 13 krpm 3 min; dry a bit in the air at RT
- resuspend with 7 µl template suppression reagent
- 90° 2 min; chill immediatly on ice
- change to special sequencing tubes and close very well with septa



Buffer change (after about 2-3 days or after more than 20 runs):
- open sequencer (only when STATUS - function is not on "collecting data")
- on the left big tube/with red line: exchange with 1 x "310 Genetic Analyzer Buffer"
- open tray with yellow TRAY button
- position 1 (left): 1 x  "310 Genetic Analyzer Buffer"  up to the black line
- position 2 (middle): water up to the black line
- position 3 (right): water, about 800 µl into opened eppendorf

Load samples:
- open sequencer (only when STATUS - function is not on "collecting data")
- open tray with yellow TRAY button
- fill tray with samples from A1 - A11 and then B2 - B12 and so on
- close sample tubes well with provided septas
- close tray, close sequencer

On the computer:
- click "310 Collection" icon
- file new; choose: sequence sample sheet 48 tube
- fill list with names of your samples
- file save
- file new; choose: sequence injection list
- choose your "sample sheet" on the new window
- module: "Seq pop6 rapid 1 ml E"
- last sample position empty with module <none>
- RUN

Generally:
- dont store labelled DNA dry (pelleted) at -20°
- dont print anything during washing/pumping/loading/..../ (because computer could crash down)
- dont open the machine while STATUS - function is on "collecting data (because run is interrupted)
- ignore: error about low gel level and error about number of runs when starting a new run