Team:EPF-Lausanne/Protocols/TetR

From 2011.igem.org

(Difference between revisions)
(PCR Cycle (30 cycles))
 
(7 intermediate revisions not shown)
Line 1: Line 1:
-
{{:Team:EPF-Lausanne/Templates/Header|title=Linear template- TetR}}
+
{{:Team:EPF-Lausanne/Templates/ProtocolHeader|title=Linear template- TetR}}
Two step extension PCR
Two step extension PCR
Line 26: Line 26:
-
<b>1.</b> 98°C for 30s
+
:<b>1.</b> 98°C for 30s
-
<b>2.</b> 98°C for 7.5 seconds
+
:<b>2.</b> 98°C for 7.5 seconds
-
<b>3.</b> 58°C for 20 sec (this depends on the primers,  
+
:<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
-
<b>4.</b> 72°C for 15 sec; you have to calculate the time required.
+
:<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)  
-
<b>5.</b> 72°C for 5 min
+
:<b>5.</b> 72°C for 5 min
-
<b>6.</b> 4°C "forever"
+
:<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
Line 41: Line 41:
== Materials ==
== Materials ==
-
    * 10µl 5x iProof HF buffer
+
* 10µl 5x iProof HF buffer
-
    * 0.5 µl of each extension primer@500nM
+
* 0.5 µl of each extension primer@500nM
-
    * 1 µl dNTP mix of 10mM
+
* 1 µl dNTP mix of 10mM
-
    * 0.5 µl High Fidelity Polymerase
+
* 0.5 µl High Fidelity Polymerase
-
    * 1 µl of gene specific PCR product from previous step
+
* 1 µl of gene specific PCR product from previous step
-
    * 0.5 µl Polymerase
+
* 0.5 µl Polymerase
-
    * 36.5 µl H2O  
+
* 36.5 µl H2O  
Extension Primers:
Extension Primers:
Line 57: Line 57:
== PCR Cycle (10 cycles) ==
== PCR Cycle (10 cycles) ==
-
<b>1.</b> 98°C for 30s
+
:<b>1.</b> 98°C for 30s
-
<b>2.</b> 98°C for 7.5 seconds
+
:<b>2.</b> 98°C for 7.5 seconds
-
<b>3.</b> 58°C for 20 sec  
+
:<b>3.</b> 58°C for 20 sec  
-
<b>4.</b> 72°C for 15 sec;  
+
:<b>4.</b> 72°C for 15 sec;  
-
(calculate for the longest fragment)
+
:<b>5.</b> 72°C for 5 min
-
<b>5.</b> 72°C for 5 min
+
:<b>6.</b> 4°C "forever"
-
<b>6.</b> 4°C "forever"
+
== Materials ==
== Materials ==
-
    * 10µl 5x iProof HF buffer
+
* 10µl 5x iProof HF buffer
-
    * 0.5 µl of each final primer @50µM/1µl of primer mix@1µM
+
* 0.5 µl of each final primer @50µM/1µl of primer mix@1µM
-
    * 1 µl dNTP mix of 10mM
+
* 1 µl dNTP mix of 10mM
-
    * 0.5 µl High Fidelity Polymerase
+
* 0.5 µl High Fidelity Polymerase
-
    * 1 µl of previous PCR product(extension PCR)
+
* 1 µl of previous PCR product(extension PCR)
-
    * 0.5 µl Polymerase
+
* 0.5 µl Polymerase
-
    * 36.5 µl H2O  
+
* 36.5 µl H2O  
Final primers
Final primers
Line 83: Line 82:
== PCR Cycle (30 cycles) ==
== PCR Cycle (30 cycles) ==
-
<b>1.</b> 98°C for 30s
+
:<b>1.</b> 98°C for 30s
-
<b>2.</b> 98°C for 7.5 seconds
+
:<b>2.</b> 98°C for 7.5 seconds
-
<b>3.</b> 53°C for 20 sec
+
:<b>3.</b> 53°C for 20 sec
-
<b>4.</b> 72°C for 15 sec;  
+
:<b>4.</b> 72°C for 15 sec;  
-
<b>5.</b> 72°C for 5 min
+
:<b>5.</b> 72°C for 5 min
-
<b>6.</b> 4°C "forever"
+
:<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