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

Line 6: Line 6:
 
<div class="wrapper">
 
<div class="wrapper">
 
<div class="header">
 
<div class="header">
−
<div class="field fw3">Ingredient</div>
+
<div class="field fw5">Ingredient</div>
 
<div class="field fw3">20 μl reaction</div>
 
<div class="field fw3">20 μl reaction</div>
−
<div class="field fw7">Final Concentration</div>
+
<div class="field fw3">Final Concentration</div>
 
</div>
 
</div>
 
<div class="record">
 
<div class="record">
−
<div class="field fw3">\( \mathrm{H_2O}\)</div>
+
<div class="field fw5">\( \mathrm{H_2O}\)</div>
 
<div class="field fw3">Add to 20 μl</div>
 
<div class="field fw3">Add to 20 μl</div>
−
<div class="field fw7"></div>
+
<div class="field fw3"></div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
−
<div class="field fw3">5x Phusion HF buffer*</div>
+
<div class="field fw5">5x Phusion HF buffer*</div>
 
<div class="field fw3">4 μl</div>
 
<div class="field fw3">4 μl</div>
−
<div class="field fw7">1x</div>
+
<div class="field fw3">1x</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
−
<div class="field fw3">10 mM dNTPs</div>
+
<div class="field fw5">10 mM dNTPs</div>
 
<div class="field fw3">0.4 μl</div>
 
<div class="field fw3">0.4 μl</div>
−
<div class="field fw7">200 μM each</div>
+
<div class="field fw3">200 μM each</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
−
<div class="field fw3">Forward primer**</div>
+
<div class="field fw5">Forward primer**</div>
 
<div class="field fw3">X μl</div>
 
<div class="field fw3">X μl</div>
−
<div class="field fw7">0.5 μM</div>
+
<div class="field fw3">0.5 μM</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
−
<div class="field fw3">Template DNA</div>
+
<div class="field fw5">Template DNA</div>
 
<div class="field fw3">X μl</div>
 
<div class="field fw3">X μl</div>
−
<div class="field fw7"></div>
+
<div class="field fw3"></div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
−
<div class="field fw3">(DMSO***, optional)</div>
+
<div class="field fw5">(DMSO***, optional)</div>
 
<div class="field fw3">(0.6 μl)</div>
 
<div class="field fw3">(0.6 μl)</div>
−
<div class="field fw7">(3%)</div>
+
<div class="field fw3">(3%)</div>
 
</div>
 
</div>
 
           <div class="record">
 
           <div class="record">
−
<div class="field fw3">Phusion DNA polymerase</div>
+
<div class="field fw5">Phusion DNA polymerase</div>
 
<div class="field fw3">0.2 μl</div>
 
<div class="field fw3">0.2 μl</div>
−
<div class="field fw7">0.02 U/ μL</div>
+
<div class="field fw3">0.02 U/ μL</div>
 
</div>
 
</div>
 
</div>
 
</div>

Revision as of 12:55, 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.