Team:EPF-Lausanne/Protocols/TetR

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(Materials)
 
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{{:Team:EPF-Lausanne/Templates/Header|title=Linear template- TetR}}
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{{:Team:EPF-Lausanne/Templates/ProtocolHeader|title=Linear template- TetR}}
Two step extension PCR
Two step extension PCR
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*<b>1.</b> 98°C for 30s
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:<b>1.</b> 98°C for 30s
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*<b>2.</b> 98°C for 7.5 seconds
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:<b>2.</b> 98°C for 7.5 seconds
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*<b>3.</b> 58°C for 20 sec (this depends on the primers,  
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:<b>3.</b> 58°C for 20 sec (this depends on the primers,  
therefore look up the annealing temperature of the primers used
therefore look up the annealing temperature of the primers used
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*<b>4.</b> 72°C for 15 sec; you have to calculate the time required.
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:<b>4.</b> 72°C for 15 sec; you have to calculate the time required.
This polymerase has an effciency of 15-30s/kb.(calculate for the longest fragment)  
This polymerase has an effciency of 15-30s/kb.(calculate for the longest fragment)  
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*<b>5.</b> 72°C for 5 min
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:<b>5.</b> 72°C for 5 min
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*<b>6.</b> 4°C "forever"
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:<b>6.</b> 4°C "forever"
*Save the file in the MAIN folder→ OK→ SAVE→RUN→Select the block you use→ RUN→ Change sample volume to 50µl, LidT should be 105°C and the box just below ("hotlid") should ALWAYS be checked
*Save the file in the MAIN folder→ OK→ SAVE→RUN→Select the block you use→ RUN→ Change sample volume to 50µl, LidT should be 105°C and the box just below ("hotlid") should ALWAYS be checked
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== PCR Cycle (10 cycles) ==
== PCR Cycle (10 cycles) ==
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<b>1.</b> 98°C for 30s
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:<b>1.</b> 98°C for 30s
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<b>2.</b> 98°C for 7.5 seconds
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:<b>2.</b> 98°C for 7.5 seconds
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<b>3.</b> 58°C for 20 sec  
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:<b>3.</b> 58°C for 20 sec  
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<b>4.</b> 72°C for 15 sec;  
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:<b>4.</b> 72°C for 15 sec;  
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(calculate for the longest fragment)
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:<b>5.</b> 72°C for 5 min
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<b>5.</b> 72°C for 5 min
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:<b>6.</b> 4°C "forever"
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<b>6.</b> 4°C "forever"
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== Materials ==
== Materials ==
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    * 10µl 5x iProof HF buffer
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* 10µl 5x iProof HF buffer
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    * 0.5 µl of each final primer @50µM/1µl of primer mix@1µM
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* 0.5 µl of each final primer @50µM/1µl of primer mix@1µM
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    * 1 µl dNTP mix of 10mM
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* 1 µl dNTP mix of 10mM
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    * 0.5 µl High Fidelity Polymerase
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* 0.5 µl High Fidelity Polymerase
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    * 1 µl of previous PCR product(extension PCR)
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* 1 µl of previous PCR product(extension PCR)
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    * 0.5 µl Polymerase
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* 0.5 µl Polymerase
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    * 36.5 µl H2O  
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* 36.5 µl H2O  
Final primers
Final primers
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== PCR Cycle (30 cycles) ==
== PCR Cycle (30 cycles) ==
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<b>1.</b> 98°C for 30s
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:<b>1.</b> 98°C for 30s
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<b>2.</b> 98°C for 7.5 seconds
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:<b>2.</b> 98°C for 7.5 seconds
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<b>3.</b> 53°C for 20 sec
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:<b>3.</b> 53°C for 20 sec
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<b>4.</b> 72°C for 15 sec;  
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:<b>4.</b> 72°C for 15 sec;  
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<b>5.</b> 72°C for 5 min
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:<b>5.</b> 72°C for 5 min
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<b>6.</b> 4°C "forever"
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:<b>6.</b> 4°C "forever"
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 08:48, 15 July 2011