Rutgers:Big Dye Sequencing

Big dye sequencing reaction

4.1- Equipment

• PCR machine

4.2- Reagents

4.3- Other consumables

4.4- Procedure

• 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

1. Add 1/10 volume of 3M sodium acetate and 2-3 volumes of 100% Ethanol
2. Mix and freeze overnight in -20.
3. Spin at full speed in a standard microcentrifuge at 4 degrees for 30 minutes.
4. Dry the pellet.
5. Add your desired quantity of water. Vortex and spin down to resuspend.

Sequencing

See Sequencing DNA

6.1- Equipment

• Applied Biosystems "3100-Avant Genetic Analyze"

6.2- Reagents

6.3- Other consumables

• sequencing plate
• septum

6.4- Procedure

• 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