3.Flick the tubes gently to mix—do not vortex. Centrifuge the tubes briefly (10 sec. at max rpm) to collect the contents at the bottom of each tube.
4. Store the dNTP/ Cy5-ddNTP mixes on ice and in the dark until needed. (Cover tubes with aluminum foil).
B. Preparation of Master Mix (Estimated Time 15-30 minutes)
NOTE: Make sure you have a water bath heating to 70ºC while mixes are being prepared. It will need to be up to temperature prior to loading samples for sequencing.
5. Label one 1.5 mL microcentrifuge tube with the DNA sample name for each DNA template with primer to be sequenced. Keep tubes on ice and capped when not in use. Ex. “DNA#1 Forward Master Mix”, “DNA #1 Reverse Master Mix”, “DNA #2 Forward Master Mix”, etc.
6. Using the table below, each group will prepare master mixes for each DNA template with primer to be sequenced. Begin by adding the water first, Reaction Buffer, DNA and primer, and Thermo Sequenase polymerase last. **Important: The Thermo Sequenase polymerase must be kept on ice and capped whenever possible. Handling time must be kept brief.
|
DNA template (0.2-0.5 µg) |
____ µL |
|
Primer (4 pmol total) |
2 µL |
|
Reaction Buffer |
4.0 µL |
|
Thermo Sequenase I DNA polymerase (10 U/ul) |
1.0 µL |
|
Distilled Water (to a final volume of 27 µL) |
____ µL |
|
Total volume |
27.0 µL |
3.Flick the tubes gently to mix—do not vortex. Centrifuge the tubes briefly (10 sec. at max rpm) and return to ice immediately.
C. Bringing the termination mixes and DNA Master Mixes together (Estimated Time 15-30 minutes)
4. Label four 0.2 mL PCR tubes for each DNA Master Mix “A, C, T, and G”. 
9.Aliquot 6 µL of the Master Mix from section B into each of the 0.2 mL PCR tubes you have just labeled, A, C, T, and G. Return tubes to ice.
10.Aliquot 2 µL of the corresponding termination mix ddA, ddC, ddG, ddT from section A into each A, C, T, G labeled 0.2 ml PCR tube. Return tubes to ice
11.Flick the tubes gently to mix—do not vortex. Centrifuge the tubes briefly (10 sec. at max rpm) to collect at the bottom of the tube and return to ice immediately.
D. Programming the Thermalcycler (Estimated Time 15-25 minutes for set up and 2.5 -3.0 hours for cycling)
12.Program and run the thermocycler (PCR machine) using the following parameters. 
