Team:SDU-Denmark/labwork/Protocols/PCR

From 2012.igem.org

(Difference between revisions)
Line 307: Line 307:
3. Annealing 55°C 30sek</br>
3. Annealing 55°C 30sek</br>
4. Elonging 72°C 1min (1kb/min) </br>
4. Elonging 72°C 1min (1kb/min) </br>
-
5. GOTO 2, x 29 cycles</br>
+
5. GO TO step 2, x 29 cycles</br>
6. End 72°C 5min</br>
6. End 72°C 5min</br>
7. Hold 4°C infinite </br>
7. Hold 4°C infinite </br>

Revision as of 17:46, 15 September 2012

iGEM TEAM ::: SDU-DENMARK courtesy of NIAID

mRNA Isolation PCR Miniprep Check Digest
Content Content Content Content
Content Content Content Content

Polymerase Chain Reaction

Proof-reading PCR using Phusion Hot Start II

some_text

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. GO TO step 2, x 29 cycles
6. End 72°C 5min
7. Hold 4°C infinite

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