Polymerase Chain Reaction
Proof-reading PCR using Phusion Hot Start II
Component 50μl reaction Final conc.
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H2O add to 50μl -
5x Phusion HF buffer 10μl 1x
10mMdNTPs 1μl 200μM each
Primer A xμl 0,5μM
Primer B xμl 0,5μM
Template DNA xμl -
(DMSO, optional) (1,5μl) (3%)
Phusion DNA Polymerase 0,5μl 0,02U/μl
PCR program:
95°C for 30 seconds
25-35 cycles:
98°C for 30 seconds
Annealing temperature (5°C below primer melting temperature)
72°C for 15-30s/kb
72°C for 5-10 minutes
4°C on hold
Non-proof-reading PCR using Taq Polymerase
5μl Dream Taq Buffer
5μl dNTP
1μl VF2
1μl VR
Udtag 10μl fra primer stock og fortynd i 90μl oprenset vand
1,5μl Dream Taq Polymerase
1μl Template
H2O up to 50μl
PCR program:
95°C for 2 minutes
29 cycles:
95°C for 1 minut
55°C for 30 sek
72°C for 1min (1kb/min)
72°C for 5 minutes
4°C on hold
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