Next Generation Sequencing and Bioinformatics
at the University of Edinburgh

Methods used by the GenePool

Sanger Sequencing: TempliPhi Protocol.

                              1 sample    1 plate 
   Sample Buffer (RED)        5 µl        500 µl 
   Reaction Buffer (BLUE)     5 µl        500 µl 
   Enzyme Mix  (YELLOW)       0.2 µl        20 µl 

DNA template 1 µl in each well

1. Dispense 62.5 µl of sample buffer to the first row of the plate and aliquot 5 µl to each well.
2. Add 1 µl of DNA template to each well
3. Heat at 95°C for 3 mins. Cool to 4°C
4. Mix 500 µl of reaction buffer and 20 µl of enzyme mix in an eppendorf. Add 65 µl to each well of a 8-well strip tube.
5. Aliquot 5 µl of the mix into each well of the plate
6. Incubate at 30°C for 4-18 hrs on a PCR machine
7. Heat at 65°C for 10mins Cool to room temperature.
8. Samples are ready to sequence directly unsing 1 µl as template.

The GenePool has core support from: GenePool admin site
The University of Edinburgh
School of Biological Sciences
The Darwin Trust
of Edinburgh

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This is version 2.2 of the GenePool website (July 2009).