Difference between revisions of "Team:Freiburg/Protocols/PCR"
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| + | <p> Polymerase Chain Reaction (PCR) is a commonly used method to amplify distinct DNA sequences. The double-stranded DNA template is denatured at high temperature. Lowering the temperature allows short oligonucleotides to anneal to the single strands at complementary regions. A DNA polymerase is used to elongate these primers complementary to the template strand by incorporation of dNTPs from 5' to 3'. After this extention step is finished, another denaturation step starts followed by primer annealing and again an extention step. By repeating this cycle numerous times the DNA fragment inbetween to primers with opposing orientation is specifically amplified.<br> | ||
| + | Further extention of the oligonucleotides allows to attach short sequences to the desired fragments. This can be used to append restriction enzyme recognition sites or Gibson Assembly suitable overhangs, for example. | ||
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Revision as of 15:34, 16 September 2015
PCR
Polymerase Chain Reaction (PCR) is a commonly used method to amplify distinct DNA sequences. The double-stranded DNA template is denatured at high temperature. Lowering the temperature allows short oligonucleotides to anneal to the single strands at complementary regions. A DNA polymerase is used to elongate these primers complementary to the template strand by incorporation of dNTPs from 5' to 3'. After this extention step is finished, another denaturation step starts followed by primer annealing and again an extention step. By repeating this cycle numerous times the DNA fragment inbetween to primers with opposing orientation is specifically amplified.
Further extention of the oligonucleotides allows to attach short sequences to the desired fragments. This can be used to append restriction enzyme recognition sites or Gibson Assembly suitable overhangs, for example.
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)
