Sequencing Reaction Protocol

Sequencing Reaction Protocol
***Do on Ice***
You will need 2 PCR tubes for each sample—one for the forward primer and one for the reverse
primer. For example, if you have 4 samples you want to sequence, you will need 8 PCR tubes.
(Numbers below are for one reaction)
1.0 µL
2.0 µL
*5X Buffer
1.75 µL
4.75 µL
*Big Dye
0.5 µL Keep this cold!
*Make a master mix: Mix buffer and H2O, then add Big Dye. You need to make
sure it is well-mixed before you add the Big Dye since it includes the polymerase.
The amounts depend on how many reactions you have.
Add primer and DNA sample to one side of the tube.
Add master mix to the other side of the tube.
Cap and spin down tubes before placing in thermocycler.
Thermocycler Profile:
96°C (0:10); 53°C (0:05); 60°C (3:00) x 33