DNA Sequencing Reaction Protocol

Quantities per reaction:

Template:

ssDNA: 50 ng
dsDNA: 200-300 ng (plasmids no larger than 10 kb)
PCR product (up to 600 bp): 30-70 ng
BAC DNA: contact the DNA Facility

Primer: 1.6 pmoles per reaction

1. Combine template, primer and water into a 0.2 ml tube. They should total 6 ul. Mix gently.

2. Add 4 ul of Big Dye Terminator Ready Reaction Mix to each tube.
3. Mix gently.
4. Spin down tubes.
5. Place tubes in thermocycler:

1. 96°C 5 min
2. 30 cycles:
   96°C 10 sec
   50°C 5 sec
   60°C 4 min
   
3. one cycle:
   4°C Hold

6. Spin down tubes.
7. Add 20 ul water to each tube.
8. Load samples on Centri·Sep Spin Column, Millipore Microcon-PCR Filter Unit (Amicon), Qiagen DyeEx Spin, or equivalent column to remove unincorporated dyes. Follow manufacture's instructions and provide the sequencing sample to the DNA Lab once it has been COMPLETELY dried down.