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

 
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<div class="header">
 
<div class="header">
 
<div class="field fw5">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 fw3">Final Concentration</div>
 
<div class="field fw3">Final Concentration</div>
 
</div>
 
</div>
 
<div class="record">
 
<div class="record">
 
<div class="field fw5">\( \mathrm{H_2O}\)</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 fw3"></div>
 
<div class="field fw3"></div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw5">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 fw3">1x</div>
 
<div class="field fw3">1x</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw5">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 fw3">200 μM each</div>
 
<div class="field fw3">200 μM each</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw5">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 fw3">0.5 μM</div>
 
<div class="field fw3">0.5 μM</div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw5">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 fw3"></div>
 
<div class="field fw3"></div>
 
</div>
 
</div>
 
         <div class="record">
 
         <div class="record">
 
<div class="field fw5">(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 fw3">(3%)</div>
 
<div class="field fw3">(3%)</div>
 
</div>
 
</div>
 
           <div class="record">
 
           <div class="record">
 
<div class="field fw5">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 fw3">0.02 U/ μL</div>
 
<div class="field fw3">0.02 U/ μL</div>
 
</div>
 
</div>
Line 110: Line 110:
 
</div>
 
</div>
 
<div class="caption">Thermal Cycling.</div>
 
<div class="caption">Thermal Cycling.</div>
−
</div>
 
−
</html>
 
−
}}
 
−
{{Team:Groningen/TEMPLATES/STEP
 
−
|title=Restriction
 
−
|content=<html>
 
−
<div class="object data" id="tbl1">
 
−
<div class="wrapper">
 
−
<div class="header">
 
−
<div class="field fw3">Component</div>
 
−
<div class="field fw3">Plasmid DNA</div>
 
−
<div class="field fw3">Unpurified Product</div>
 
−
                <div class="field fw3">Genomic DNA</div>
 
−
</div>
 
−
<div class="record">
 
−
<div class="field fw3">Water, nuclease free*</div>
 
−
<div class="field fw3">up to 20 μl</div>
 
−
<div class="field fw3">up to 20 μl</div>
 
−
                <div class="field fw3">up to 50 μl</div>
 
−
</div>
 
−
<div class="record">
 
−
<div class="field fw3">10x NEB 2.1 buffer</div>
 
−
<div class="field fw3">2 μl</div>
 
−
<div class="field fw3">2 μl**</div>
 
−
                <div class="field fw3">5 μl</div>
 
−
</div>
 
−
<div class="record">
 
−
<div class="field fw3">DNA</div>
 
−
<div class="field fw3">up to 1 μg</div>
 
−
<div class="field fw3">up to 0.2 μg</div>
 
−
                <div class="field fw3">up to 5 μg</div>
 
−
</div>
 
−
<div class="record">
 
−
<div class="field fw3">NEB Enzyme</div>
 
−
<div class="field fw3">0.5 μl</div>
 
−
<div class="field fw3">0.5 μl</div>
 
−
                <div class="field fw3">0.5 μl</div>
 
−
</div>
 
−
        <div class="record">
 
−
<div class="field fw3">Total Volume</div>
 
−
<div class="field fw3">20 μl</div>
 
−
<div class="field fw3">20 μl</div>
 
−
                <div class="field fw3">20 μl</div>
 
−
</div>
 
−
</div>
 
−
<div class="caption">Restriction.</div>
 
 
</div>
 
</div>
 
</html>
 
</html>
 
}}
 
}}

Latest revision as of 21:50, 21 November 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.