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In general, we use primer sequencing for all first pass sequencing attempts. Terminator sequencing is used
when first pass sequencing reveals an indel. (See Polymerase Chain Reaction Protocol.)
Our experiments have shown that the best way to prepare out PCR samples for sequencing is to dilute them
with water.
Our PCR reaction (see Polymerase Chain Reaction protocol) does not call for a huge excess of primers or
dNTPs. Instead of removing the excess primers and dNTPS or breaking them down enzymatically, we have
found that diluting with water is cheaper, easier and faster.
Equipment
DNA Engine Tetrad (MJ Research PTC-225)
Jouan Centrifuge (CR422)
Multimek 96 Automated 96-Channel Pipettor (Beckman Instruments)
384-split-well plate (Cat. No. 1055-40-0 from Robbins Scientific)
Reagents
0.01% Triton H2O
Biosystems Big Dye Primer Forward 5000-reaction kit (Cat. No. 403049)
Applied Biosystems Big Dye Primer Reverse 5000-reaction kit (Cat. No. 403050)
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11/13/2019 Big Dye Primer Sequencing Reaction Protocol
melt these frozen bits by running one's hand over the foil and then spinning the plate down
before removing the foil.
2. Store these plates on ice until the next step (see below) is completed. Preheat two Tetrad alpha blocks.
3. Initialize the robot and open the program called Insert. This program mixes the PCR product 25 times
(to mix the PCR product thoroughly with the triton water). When the mixing the robot pauses, take the
sequencing plates off the ice and place them on the robot deck.
4. Start the program again. The robot will add 1 L of the diluted PCR product to each of the four
quadrants on the forward plate. This process will be repeated for the reverse plate.
5. 6. After the PCR product has been added to the pre-made sequencing plates, spin them down in the
Jouan and cover them with Cycle Seal PCR Plate Sealer. Put the reactions in the Tetrad, set the
reaction volume to 12 µL, and run the following program:
BIGPRIME
1. 96° C for 10 sec
2. 55° C for 5 sec
3. 70° C for 1 min
4. Repeat steps 1-3 14 more times
5. 96° C for 10 sec
6. 70° C for 1 min
7. Repeat 4-5 14 more times
8. Go to 4° C
When the reaction is done (it takes about 1.5 hours) remove it from the Tetrad and store it in the fridge until
the precipitation can be started (see Ethanol Precipitation Protocol).
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