Polymerase Chain Reaction
Proof-reading PCR using Phusion Hot Start II
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:
1. Start 95°C 2min
2. Denaturing 95°C 1min
3. Annealing 55°C 30sek
4. Elonging 72°C 1min (1kb/min)
5. GOTO 2, x 29 cycles
6. End 72°C 5min
7. Hold 4°C infinite
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