Team:EPF-Lausanne/Protocols/1.5 steps PCR

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<font size="6" color="#007CBC"><i>Miniprep Protocol</i></font>  
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<font size="6" color="#007CBC">1.5 Steps PCR Protocol</font>  
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'''1.''' Do the following mixing:
'''1.''' Do the following mixing:
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{| class="wikitable" style="text-align:center; width:30%;"
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<center>
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{| class="wikitable" style="text-align:left; width:30%;"
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! scope=row | dNTP
! scope=row | dNTP
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|'''50 μl'''
|'''50 μl'''
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</center>
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'''2.''' Make it run '''10''' cycles:
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<center>
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{| class="wikitable" style="text-align:left; width:20%;"
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|-
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! scope=row | 1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|94°C
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|4:00
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|-
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! scope=row | 2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|94°C
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|0:30
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|-
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! scope=row | 3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|55°C
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|1:00
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|-
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! scope=row | 4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|72°C
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|2:00
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|-
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! scope=row | 5&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|Cycle 2-4 10 times
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|
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|-
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! scope=row | 6&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|72°C
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|7:00
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|-
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! scope=row | 7&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|4°C
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|&infin;
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|}
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</center>
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'''3.''' We now add the standard iGEM primers which finish the extension, and do the following cycles:
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<center>
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{| class="wikitable" style="text-align:left; width:20%;"
 +
|-
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! scope=row | 1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|94°C
 +
|4:00
 +
|-
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! scope=row | 2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|94°C
 +
|0:30
 +
|-
 +
! scope=row | 3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|55°C
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|1:00
 +
|-
 +
! scope=row | 4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
|72°C
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|2:00
 +
|-
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! scope=row | 5&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|Cycle 2-4 25-30 times
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|
 +
|-
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! scope=row | 6&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|72°C
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|7:00
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|-
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! scope=row | 7&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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|4°C
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|&infin;
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|}
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</center>
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Latest revision as of 18:44, 20 October 2009



1.5 Steps PCR Protocol



1. Do the following mixing:

dNTP 1 μl
5x Buffer + MgCl2 10 μl
Primer Mix 1 μl
template 0.5 μl
HiFi Plus DNAp 0.5 μl
dH2O 36.5 μl
Final Volume 50 μl


2. Make it run 10 cycles:

1             94°C 4:00
2             94°C 0:30
3             55°C 1:00
4             72°C 2:00
5             Cycle 2-4 10 times
6             72°C 7:00
7             4°C ∞


3. We now add the standard iGEM primers which finish the extension, and do the following cycles:

1             94°C 4:00
2             94°C 0:30
3             55°C 1:00
4             72°C 2:00
5             Cycle 2-4 25-30 times
6             72°C 7:00
7             4°C ∞


Retrieved from "http://2009.igem.org/Team:EPF-Lausanne/Protocols/1.5_steps_PCR"