Team:EPF-Lausanne/Protocols/TetR

From 2011.igem.org

(Difference between revisions)
(PCR Cycle (10 cycles))
 
(15 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
*Press STATUS to check the progress
*Press STATUS to check the progress
-
 
== <b>Extension PCR</b> ==
== <b>Extension PCR</b> ==
== Materials ==
== Materials ==
-
 
+
* 10µl 5x iProof HF buffer
-
* Gene PCR product
+
* 0.5 µl of each extension primer@500nM
-
* Extension primers 500nM
+
* 1 µl dNTP mix of 10mM
 +
* 0.5 µl High Fidelity Polymerase
 +
* 1 µl of gene specific PCR product from previous step
 +
* 0.5 µl Polymerase
 +
* 36.5 µl H2O
Extension Primers:
Extension Primers:
Line 52: Line 55:
Reverse: CAA AAA ACC CCT CAA GAC CCG TTT AGA GGC CCC AAG GGG TTA TGC TAG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTA GCA GCC TGA GTC G; <b>Tm=70.4ºC</b>
Reverse: CAA AAA ACC CCT CAA GAC CCG TTT AGA GGC CCC AAG GGG TTA TGC TAG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTA GCA GCC TGA GTC G; <b>Tm=70.4ºC</b>
-
== PCR Cycle (30 cycles) ==
+
== PCR Cycle (10 cycles) ==
-
 
+
-
<b>1.</b> 98°C for 30s
+
-
<b>2.</b> 98°C for 7.5 seconds
+
-
<b>3.</b> 58°C for 20 sec
+
-
<b>4.</b> 72°C for 15 sec;
+
-
(calculate for the longest fragment)
+
-
<b>5.</b> 72°C for 5 min
+
-
<b>6.</b> 4°C "forever"
+
 +
:<b>1.</b> 98°C for 30s
 +
:<b>2.</b> 98°C for 7.5 seconds
 +
:<b>3.</b> 58°C for 20 sec
 +
:<b>4.</b> 72°C for 15 sec;
 +
:<b>5.</b> 72°C for 5 min
 +
:<b>6.</b> 4°C "forever"
== Materials ==
== Materials ==
-
* 1µl Final primers
+
* 10µl 5x iProof HF buffer
 +
* 0.5 µl of each final primer @50µM/1µl of primer mix@1µM
 +
* 1 µl dNTP mix of 10mM
 +
* 0.5 µl High Fidelity Polymerase
 +
* 1 µl of previous PCR product(extension PCR)
 +
* 0.5 µl Polymerase
 +
* 36.5 µl H2O
Final primers
Final primers
Line 73: Line 80:
Reverse: CAA AAA ACC CCT CAA GAC; <b>Tm=48.6ºC </b>
Reverse: CAA AAA ACC CCT CAA GAC; <b>Tm=48.6ºC </b>
-
== PCR Cycle (10 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> 58°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