mohammad tabish ahmed - eTheses Repository - University of ...

Chapter 2
- Colony/culture 1μl
- SDW made up to 50μl
The samples were PCR-amplified in a Eppendorf vapo.protect thermocycler under the
following conditions:
Segment Cycles Temperature Time
1 1 94°C 4 minutes
94°C 45 seconds
2 30
55°C 1 minute
72°C 1-5 minutes
3 1 72°C 10 minutes
Alternatively, the Thermo Scientific ReadyMix master mix was also used (23μl with 0.5μl of
each primer and 1μl culture). The cycling conditions were the same as that listed above.
Site directed mutagenesis
The QuickChange Site-Directed Mutagenesis Kit (Agilent, formerly Stratagene) was used as
per the manufacturer’s instructions. The PCR protocol used is as follows:
- 10X reaction buffer 5μl
- DNA 50ng of dsDNA template
- Primers 12.5pmole (for each primer)
- dNTP mix 0.8 mM (final concentration)
- SDW made up to 50μl
- PfuTurbo/PfuUltra DNA polymerase 1μl
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