Rutgers:Big Dye Sequencing
Big dye sequencing reaction
- PCR machine
4.3- Other consumables
- All the following steps must be made on ice
- Prepare the following mix (quantities are for 1 tube):
- enhancer: 1 µl (if sequence GC rich) - big dye: 1 µl - primer M13: 0.33 µl (10 µM stock=3.3 pmol) - 5X buffer: 1.0 µl - water: 4.67 µl - cleaned template: 2 µl
- put 8 µl of the mix in strip tubes
- add 2 µl of template
- Use a PCR machine for cycle sequecing (SWATI-SEQQ):
lid temp: 110.0°C 96°C: 10 sec 50°C: 5 sec 60°C: 4 min 25°C: pause
Cleaning of the sequencing reaction products by precipitation
Best protocols would be under Purification of DNA.
General Procedure goes as follows
- Add 1/10 volume of 3M sodium acetate and 2-3 volumes of 100% Ethanol
- Mix and freeze overnight in -20.
- Spin at full speed in a standard microcentrifuge at 4 degrees for 30 minutes.
- Dry the pellet.
- Add your desired quantity of water. Vortex and spin down to resuspend.
See Sequencing DNA
- Applied Biosystems "3100-Avant Genetic Analyze"
6.3- Other consumables
- sequencing plate
- launch "3100 Avant data coll"
- ignore request for ZIP disk
- insert sample names (BLOCKS of four)
- Dye set: "z"
- Mobility file: DT3100POP6(BDv3)v1.mob
- Project name: 3100-Avant project1
- Run module: XXXXLongRun
- Analysis module: BC-3100APOP6SR_SeqOffFtOff.saz
- Click OK
- place the plate on a base and a cover on top, press down
- press "Tray"
- put plate, press evenly down, close door
- select "JPG", click plate image (goes from yellow to green)
- click on the green triangle pointing to the right
- go to status or run view