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

Line 55: Line 55:
 
<div class="field fw3">Cycle Step</div>
 
<div class="field fw3">Cycle Step</div>
 
<div class="field fw3">Temperature</div>
 
<div class="field fw3">Temperature</div>
−
<div class="field fw7">Time</div>
+
<div class="field fw3">Time</div>
−
                 <div class="field fw7">Cycle</div>
+
                 <div class="field fw3">Cycle</div>
  
 
</div>
 
</div>
Line 62: Line 62:
 
<div class="field fw3">Initial denaturation</div>
 
<div class="field fw3">Initial denaturation</div>
 
<div class="field fw3">98 °C</div>
 
<div class="field fw3">98 °C</div>
−
<div class="field fw7">30 s</div>
+
<div class="field fw3">30 s</div>
−
                 <div class="field fw7">1</div>
+
                 <div class="field fw3">1</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw3">Denaturation</div>
 
<div class="field fw3">Denaturation</div>
 
<div class="field fw3">98 °C</div>
 
<div class="field fw3">98 °C</div>
−
<div class="field fw7">5-10 s</div>
+
<div class="field fw3">5-10 s</div>
−
                 <div class="field fw7">25-35</div>
+
                 <div class="field fw3">25-35</div>
 
</div>
 
</div>
 
                 <div class="record">
 
                 <div class="record">
 
<div class="field fw3">Annealing</div>
 
<div class="field fw3">Annealing</div>
 
<div class="field fw3">T m - 5°C</div>
 
<div class="field fw3">T m - 5°C</div>
−
<div class="field fw7">10-30 s</div>
+
<div class="field fw3">10-30 s</div>
−
                 <div class="field fw7">25-35</div>
+
                 <div class="field fw3">25-35</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw3">Extension</div>
 
<div class="field fw3">Extension</div>
 
<div class="field fw3">72 °C</div>
 
<div class="field fw3">72 °C</div>
−
<div class="field fw7">15-30 s/kb</div>
+
<div class="field fw3">15-30 s/kb</div>
−
                 <div class="field fw7">25-35</div>
+
                 <div class="field fw3">25-35</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw3">Final extension</div>
 
<div class="field fw3">Final extension</div>
 
<div class="field fw3">72°C</div>
 
<div class="field fw3">72°C</div>
−
<div class="field fw7">5-10 min</div>
+
<div class="field fw3">5-10 min</div>
−
                 <div class="field fw7">1</div>
+
                 <div class="field fw3">1</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw3">Hold</div>
 
<div class="field fw3">Hold</div>
 
<div class="field fw3">4 °C</div>
 
<div class="field fw3">4 °C</div>
−
<div class="field fw7">Hold</div>
+
<div class="field fw3">Hold</div>
−
                 <div class="field fw7">1</div>
+
                 <div class="field fw3">1</div>
 
</div>
 
</div>
 
</div>
 
</div>

Revision as of 12:56, 22 August 2015

PCR mix

1. 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.
2. Gently vortex the samples and spin down.
3. 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.