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

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<div class="caption">Thermal Cycling.</div>
 
<div class="caption">Thermal Cycling.</div>
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{{Team:Groningen/TEMPLATES/STEP
 +
|title=Restriction
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|content=<html>
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<div class="object data" id="tbl1">
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<div class="wrapper">
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<div class="header">
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<div class="field fw3">Component</div>
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<div class="field fw3">Plasmid DNA</div>
 +
<div class="field fw3">Unpurified Product</div>
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                <div class="field fw3">Genomic DNA</div>
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</div>
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<div class="record">
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<div class="field fw3">Water, nuclease free*</div>
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<div class="field fw3">up to 20 μl</div>
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<div class="field fw3">up to 20 μl</div>
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                <div class="field fw3">up to 50 μl</div>
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</div>
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<div class="record">
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<div class="field fw3">10x NEB 2.1 buffer</div>
 +
<div class="field fw3">2 μl</div>
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<div class="field fw3">2 μl**</div>
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                <div class="field fw3">5 μl</div>
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</div>
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<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>
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</div>
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<div class="caption">Restriction.</div>
 
</div>
 
</div>
 
</html>
 
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Revision as of 13:35, 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.

Restriction

Component
Plasmid DNA
Unpurified Product
Genomic DNA
Water, nuclease free*
up to 20 μl
up to 20 μl
up to 50 μl
10x NEB 2.1 buffer
2 μl
2 μl**
5 μl
DNA
up to 1 μg
up to 0.2 μg
up to 5 μg
NEB Enzyme
0.5 μl
0.5 μl
0.5 μl
Total Volume
20 μl
20 μl
20 μl
Restriction.