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

Chapter 2
The PCR cycling parameters used were as follows:
Segment Cycles Temperature Time
1 1 94°C 30 seconds
94°C 45 seconds
2 30
55°C 1 minute
72°C 1 minute/Kb
3 1 72°C 10 minutes
Following PCR, the samples were put on ice for 2 minutes to cool the reaction to ≤ 37°C. 1µl
of Dpn I was added and the reaction was incubated at 37°C for 1 hour to allow the enzyme to
digest the parental DNA plasmid. The digested product was then transformed into DH5α and
plated out onto LB plates (with appropriate additives).
PCR in the construction of chimeric proteins
In chapter 4, various chimeric proteins were constructed by using a modified PCR extension
technique. The details of the process have been outlined in the experimental design of chapter
4. The DNA polymerase and buffers for these reactions were from Finnzymes. The lists of
additives in the PCR mix and the cycles have been listed below:
- 5X Phusion HF buffer 10μl
- 10 mM dNTPs 1μl
- Primer A 0.5 mM
- Primer B 0.5 mM
- Template DNA 20ng
- DMSO 1.5μl
- Phusion DNA Polymerase 0.5-1μl
- SDW Made up to 50μl
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