Team:Freiburg/Protocols/PCR
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PCR
Standard PCR Programm
For amplification of different DNA fragments from plasmid templates, various PCR approaches were used. In general, PCRs were performed with Phusion High Fidelity DNA Polymerase (NEB). The components of the reaction mixture were used in defined amounts/concentrations recommended by the manufacturer.
PCR mix
Primers for DNA amplification were designed to have a melting temperature of about 60°C (detailed information can be found in the Oligo list). The following cycling conditions have been used for standard PCRs. Extention times were calculated according to NEB's recommendations for Phusion High-Fidelity Polymerase.
Lid temperature: 98°C
98°C | 5 min | Initial denaturation |
98°C | 30 s | Denaturation |
---|---|---|
63°C | 30 s | Annealing |
72°C | 30 - 40 s pro kb | Extention |
72°C | 10 min | Final extention |
4°C | hold |
18 - 25 Zyklen (Schritt 2-4)