- Incubate 3ul PCR reaction to be sequenced with 1ul ExoSAP (exonuclease – shrimp alkaline phosphatase, GE-Amersham) for 15’ at 37ºC. We do this in duplicate for one forward and one reverse reaction.
- Inactivate the enzyme 15’ at 80ºC.
- Add 2 ul of 1-2 uM primer (forward or reverse), 2 uL of 5x Applied Biosystems BigDye buffer (blue top), 1 ul of ABI-supplied BigDye mix to each tube, and bring the final volume to 10 ul with ddH2O (generally 1uL). We use versions 1.1 and 3.1 indifferently.
- Centrifuge briefly and cycle using the terminator program (i.e. preheat at 96ºC followed by 28-30 cycles of [96ºC for 20 seconds, 55-60ºC (annealing temp for PCR) for 30 seconds, 60ºC for 90 seconds], and then link to a 15ºC hold).
- Proceed with the spin column purification using G-50 microtiter plate procedures given below.
a. Add dry Sephadex G-50 to the Millipore (cat.# MACL 096 45) 45 ul Column Loader. Only fill as many wells as needed to purify reactions, recover excess on clean foil.
b. Remove the excess of resin from the top of the Column Loader with the scraper supplied.
c. Place MultiScreen HV Plate (Millipore MAHVN4550) upside-down on top of the Column Loader.
d. Invert both MultiScreen HV Plate and Column Loader. and tap on top of the Millipore Column Loader to release the resin.
e. Using a multi-channel pipettor, add 300 ul of ddH2O to each well to swell the resin. Let stand at room temperature for 1-3 hours.
f. Once the minicolumns are swollen in MultiScreen plates, they can be sealed with saran wrap and stored in the refrigerator at 4 deg C for several days. A batch of plates also can be stored in the refrigerator at 4 deg C for several weeks in a sealed plastic container with a damp towel to assure the plates are kept moist.
- Spin through excess liquid over a reserved collection 96 well plate by taping the column plate to the collection plate and centrifuging for 5’ at 2000 rpm.
- ADD 20 ul ddH20 to each reaction BEFORE applying with a multichannel pipette to columns. Align over clean collection tubes and balance plate with collection tubes at other end. Spin 5’ at 2500 rpm and recover approx. 20 ul. Freeze reactions if you can not sequence that day.
- Run sequences as they are on the automated sequencer (Applied Biosystems).