Big-Dye Automated Sequencing

Reaction Mixes

10 ul Reaction:

  • 2 ul Big Dye Reaction Buffer
  • 2 ul Big Dye Terminator
  • 2 ul 10 uM primer
  • 2 ul Qiagen miniprep (200-500 ng template)


PCR Cycle (for PE 9600 or equivalent):

95oC for 2 minutes

95oC for 30 seconds
50oC for 15 seconds
60oC for 4 minutes
  ==> 30 cycles
4oC hold

Note: Ramping time is very important; should not be slower than 1 degree/second.