Difference between revisions of "Template:Team:Groningen/CONTENT/PROTOCOLS/PCR"

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|title=PCR mix
 
|title=PCR mix
 
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<div class="text">1. Create the following mix.</div>
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<div class="text">Create the following mix.</div>
 
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|title=Vortex
 
|title=Vortex
 
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<div class="text">2. Gently vortex the samples and spin down.</div>
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<div class="text">Gently vortex the samples and spin down.</div>
 
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</html>
 
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<div class="text">3. Place the reactions in a thermal cycler. Perform PCR using recommended thermal cycling conditions. </div>
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<div class="text">Place the reactions in a thermal cycler. Perform PCR using recommended thermal cycling conditions. </div>
 
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         <div class="record">
 
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<div class="field fw3">Final extension</div>
 
<div class="field fw3">Final extension</div>
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<div class="field fw3">72°C</div>
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<div class="field fw3">72 °C</div>
 
<div class="field fw3">5-10 min</div>
 
<div class="field fw3">5-10 min</div>
 
                 <div class="field fw3">1</div>
 
                 <div class="field fw3">1</div>

Revision as of 13:27, 22 August 2015

PCR mix

Create the following mix.
Ingredient
20 μl reaction
Final Concentration
\( \mathrm{H_2O}\)
Add to 20 μl
5x Phusion HF buffer*
4 μl
1x
10 mM dNTPs
0.4 μl
200 μM each
Forward primer**
X μl
0.5 μM
Template DNA
X μl
(DMSO***, optional)
(0.6 μl)
(3%)
Phusion DNA polymerase
0.2 μl
0.02 U/ μL
PCR Mix.

Vortex

Gently vortex the samples and spin down.

Thermal Cycle

Place the reactions in a thermal cycler. Perform PCR using recommended thermal cycling conditions.
Cycle Step
Temperature
Time
Cycle
Initial denaturation
98 °C
30 s
1
Denaturation
98 °C
5-10 s
25-35
Annealing
T m - 5°C
10-30 s
25-35
Extension
72 °C
15-30 s/kb
25-35
Final extension
72 °C
5-10 min
1
Hold
4 °C
Hold
1
Thermal Cycling.