BigDye Labeling Reaction Conditions

Template DNA* *Either 200 ng plasmid or 10-20 ng PCR product for every 200 bp sequence
Primer 10pmol
Big Dye Terminator V3.1 1 ul
5x Sequencing Buffer

3 ul

Total Volume 10 ul
Standard Thermal Cycling Conditions
96° C 5min. 1 cycle
96° C 15 sec. 25 cycles
50° C 15 sec.
60° C 4 min.
4° C hold
Difficult Template Thermal Cycling Conditions**
98° C 10 min. 1 cycle
98° C 30 sec. 30 cycles
50° C 30 sec.
60° C 4 min.
4° C hold  

** For difficult to sequencing templates (GC rich or hairpins), DMSO must be added at 5% final concentration to the BigDye labeling reaction.

 

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