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>
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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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Latest revision as of 07:05, 20 November 2015

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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

ingredient
volume
Template
200 - 300 ng
Primer (10 µM)
1 µl each
Q5 Polymerase
0.5 µl
dNTPs
4 µl
DMSO
1 µl
Q5 reaction buffer (5x)
10 µl
dH2O
up to 50 µl
Add GC enhancer if the GC content of the template [??] is very high.

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)