Get a printable version of this protocol (requires Adobe Acrobat)
Preparation of samples to be sequenced:
Denature DNA
Incubate at room temp. For 5 min.
Ethanol precipitate to pellet the DNA
spin for 15 min., wash with 70% EtOH, spin and speed vac to dry.
Add 7uL dH2O to the pellet to resuspend.
Heat the DNA at 95ºC for 5 min.
Quick cool by placing in ice water.
Annealing DNA to primer
Incubate at 42ºC for 20 minutes.
Labelling Reaction: (For 4 reactions)
Diluted Labelling Mix (1:10) (dITP)
Diluted sequenase (1:7)
Incubate at room temperature for 5 min.
Add 2.5uL each of G,A,T and C termination into separate tubes. (G,A,T and C that go with dITP labelling mix).
Add 3.5uL of the labelling reaction to each of the tubes with G, A, T, and C.
Incubate at 37ºC for 10 min.
Add 4uL of stop mix to each tube.
Heat samples at 85ºC for 5 min. before loading onto the gel. (Gel was pre-run by Andrew and made by Andrew)
-gel was dried at 80ºC for 1.25 hours and placed on film for overnight exposure.
Get a printable version of this protocol