Next Generation Sequencing and Bioinformatics
at the University of Edinburgh

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Methods used by the GenePool

Sanger Sequencing: PCR

Reaction volumes

                          per sample (20 µl)  per plate    2 plates         3 plates
   10X buffer (1.5mM MgCl2)    2 µl           200 µl        400 µl           600 µl
   Primer F (10pmol/ µl)     0.4 µl            40 µl         80 µl           120 µl
   Primer R (10pmol/ µl)     0.4 µl            40 µl         80 µl           120 µl
   dntps (2mM)                 2 µl           200 µl        400 µl           600 µl
   Taq (5 U/ µl)             0.2 µl            15 µl         30 µl            45 µl
   dH2O                       13 µl          1305 µl       2610 µl          3915 µl

DNA template 2 µl in each

Cycle:
   94°C    3mins
   94°C    15 sec}
   55°C    40sec } - 35 cycles
   72°C    3 min }
   72°C    10min
   hold at 4°C

1. Make up a master mix and dispense 225 µl into each well of an 8-strip tube.
For a plate dispense 110 µl in rows 1 and 7.
2. Pipette 18 µl from the strip tube into each column of the PCR plate.
3. Add 2 µl of template to each well.
4. Seal the plate and place on the PCR machine.