The iGEM Team of University of Southern Denmark 2012 igem.sdu.2012@gmail.com

mRNA Isolation	PCR	Miniprep	Check Digest
3A-Assembly	Colony-PCR	Transformation	Gel-electrophoresis
Reverse Transcriptase	Mutagenisis	PCR-,gel clean-up	Content

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:

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