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	Mutagenesis	PCR-,gel clean-up

Polymerase Chain Reaction

Proof-reading PCR using Phusion Hot Start II

Component                 50μl reaction               Final conc.

-------------------------------------------------------------------
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