Difference between revisions of "Team:EPF Lausanne/Notebook/Protocols"

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                 <li><a href="#miniprep">Miniprep</a></li>
 
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                 <li><a href="#pcr">Polymerase Chain Reaction (PCR)</a></li>
 
                 <li><a href="#pcr">Polymerase Chain Reaction (PCR)</a></li>
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                   <a class="dropdown-toggle" data-toggle="dropdown" href="#">Section 4 <span class="caret"></span></a>
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                   <a class="dropdown-toggle" data-toggle="dropdown" href="#">Polymerase Chain Reaction (PCR)<span class="caret"></span></a>
 
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                     <li><a href="#section41">Section 4-1</a></li>
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                     <li><a href="#phusionpcr">Phusion PCR</a></li>
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                     <li><a href="#section42">Section 4-2</a></li>                     
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                     <li><a href="#taqpcr">Taq PCR</a></li>                     
 
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    <!-- COLONY PCR -->
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<!-- COLONY PCR -->
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              <div id="colonypcr" class="well">         
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<div id="colonypcr" class="well">         
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                <h1>Colony PCR - based on NEB Taq PCR Protocol</h1>
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<h1>Colony PCR</h1>
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<h2>Materials</h2>
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<h2>Materials</h2>
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<p> • 10X Standard Taq Reaction Buffer</p>
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<p> • Materials for Taq PCR with petri dish with transformed colonies instead of template plsmid DNA</p>
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<p> • dNTPs</p>
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<h2>Procedure</h2>
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<p> • Taq polymerase</p>
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<p> • Prepare 25 μL reactions as in Taq PCR Protocol without template DNA</p>
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<p> • Forward and Reverse Primers</p>
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            <p> • With a sterile tip, under the flame, scrape part of a single colony and add to PCR tube</p>
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<p> • Nuclease Free Water</p>
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<p> • Mix by pipetting up and down or flicking the reactions
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<p> • Petri dish with transformed colonies</p>
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<p> • Put tubes in thermocycler with cycling conditions as described in Taq PCR Protocol</p>
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<h2>Procedure</h2>
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</div>
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<p> • Prepare following reaction in 0.5 mL PCR tubes on ice by making a Master Mix for all reactions and adding polymerase last:</p>
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<table width="100%">
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<tr>
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<th>Component</th>
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<th>25 μL reaction</th>
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<th>Final concentration</th>
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</tr>
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<tr>
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<td>10X Standard Taq Reaction Buffer</td>
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<td>2.5 μL</td>
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<td>1X</td>
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</tr>
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<tr>
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<td>10 mM dNTPs</td>
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<td>0.5 μL</td>
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<td>200 μM</td>
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</tr>
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<tr>
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<td>10 mM Forward Primer</td>
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<td>0.5 μL</td>
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<td>0.2 μM</td>
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</tr>
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<tr>
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<td>10 mM Reverse Primer</td>
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<td>0.5 μL</td>
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<td>0.2 μM</td>
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</tr>
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<tr>
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<td>Taq DNA Polymerase</td>
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<td>0.125 μL</td>
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<td>0.625 units/25 μL PCR</td>
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</tr>
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<tr>
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<td>Nuclease Free Water</td>
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<td>to 25 μL</td>
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<td>  </td>
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</tr>
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</table>
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                <p> • With a sterile tip, under the flame, scrape part of a single colony and add to PCR tube</p>
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−
<p> • Mix by pipetting up and down or flicking the reactions
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<p> • Put tubes in thermocycler with following cycling conditions:
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<table width="100%">
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<tr>
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<th>Step</th>
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<th>  </th>
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<th>Temperature</th>
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<th>Time</th>
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</tr>
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<tr>
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<td>Initial Denaturation</td>
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<td>  </td>
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<td>95°C</td>
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<td>30 seconds</td>
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</tr>
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<tr>
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<td>25 – 35 cycles</td>
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<td>Denaturation</td>
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<td>95°C</td>
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<td>15 – 30 seconds</td>
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</tr>
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<tr>
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<td>  </td>
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<td>Annealing</td>
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<td>45 – 68°C</td>
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<td>15 - 60seconds</td>
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</tr>
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<tr>
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<td>  </td>
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<td>Extension</td>
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<td>68°C</td>
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<td>1 minutes per kb</td>
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</tr>
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<tr>
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<td>Final Extension</td>
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<td>  </td>
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<td>68°C</td>
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<td>5 minutes</td>
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</tr>
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<tr>
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<td>Hold</td>
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<td>  </td>
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<td>4°C </td>
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<td>  </td>
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</tr>
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</table>
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              </div>
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     <!-- GIBSON ASSEMBLY -->
 
     <!-- GIBSON ASSEMBLY -->
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<h1>Polymerase Chain Reaction (PCR)</h1>
 
 
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<!-- PHUSION PCR -->
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<div id="phusionpcr" class="well">               
 
<h1>Phusion PCR</h1>
 
<h1>Phusion PCR</h1>
 
<h2>Materials</h2>
 
<h2>Materials</h2>
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<h2>Guidelines</h2>
 
<h2>Guidelines</h2>
 
<p>To be completed</p>
 
<p>To be completed</p>
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<!-- TAQ PCR -->
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<div id="taqpcr" class="well">
 
<h1>Taq PCR</h1>
 
<h1>Taq PCR</h1>
 
<h2>Materials</h2>
 
<h2>Materials</h2>
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                <h1>Section 4-1</h1>
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                <p>Try to scroll this section and look at the navigation list while scrolling!</p>
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              </div>     
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              <div id="section42" class="well">       
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                <h1>Section 4-2</h1>
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                <p>Try to scroll this section and look at the navigation list while scrolling!</p>
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Revision as of 09:41, 22 July 2015

Protocols

Agarose Gel

Materials

• 1X TAE

• Agarose

• Gel Red

• DNA samples

• 6X loading dye

• Nuclease free water

Procedure

Prepare 1.2% agarose gel for small fragments and 3% agarose gel for large fragments

• Mix 50 mL 1X TAE and 0.6 g (1.2%) or 1.5 g (3%) agarose

• Melt in microwave until agarose has melted (about 50 seconds)

• Add 1.3 μL (1.2%) or 1.5 μL (3%) Gel Red

• Pour solution into agarose gel mold with comb

• Let set for 20 minutes or until solid

• Place gel in 1X TAE and remove comb

• Load samples of 200 ng (or 2 μL) DNA mixed with 2 μL 6X loading dye and nuclease free water up to 12 μL

• Run gel at 100-120 Volts for 40-50 minutes (1.2%) or 80 Volts for 2 hours (3%)

• Take a picture of the gel at the UV detector

Amino acid solution

Materials

• Histidine-Hcl

• Uracil

• Leucine

• Tryptophan

Procedure

Stock concentration Final concentration Total quantity for 50 mL
100 mM Histidine-Hcl (209 g/mol) 20.9 g/L 0.418 g
20 mM Uracil (112 g/mol) 2.24 g/L 0.0448 g
100 mM Leucine (131 g/mol) 13.1 g/L 0.262 g
40 mM Tryptophan (204 g/mol) 8.16 g/L 0.1632 g

• Filter and sterilize solutions

• Add 8 mL per liter of selective medium or spread 500 μL on a selective plate

Colony PCR

Materials

• Materials for Taq PCR with petri dish with transformed colonies instead of template plsmid DNA

Procedure

• Prepare 25 μL reactions as in Taq PCR Protocol without template DNA

• With a sterile tip, under the flame, scrape part of a single colony and add to PCR tube

• Mix by pipetting up and down or flicking the reactions

• Put tubes in thermocycler with cycling conditions as described in Taq PCR Protocol

Gibson Assembly - based on NEB Gibson Assembly Protocol

Materials

• DNA fragments

• 2X Gibson Assembly Mater Mix (NEB)

• 2X NEBuilder Positive Control (NEB)

• Deionized water

Procedure

• Set up following reactions on ice, adding Gibson Assembly Master Mix last:

Component 2 – 3 Fragments Assembly 4 – 6 Fragments Assembly Positive Control
Total Amount of Fragments 0.02 – 0.5 pmols 0.2 – 1 pmols 10 μL
2X Gibson Assembly Master Mix 10 μL 10 μL 10 μL
Deionized water to 20 μL to 20 μL 0 μL

Optimized efficiency for 50 – 100 ng of vectors and 2 – 3 fold of excess inserts

• Incubate samples at 50°C for 15 minutes (2 – 3 fragments) or for 60 minutes (4 – 6 fragments)

• Store samples on ice or at -20°C until transformation

• Transform competent cells following the Transformation Protocol

Miniprep - with QIAprep Spin Miniprep Kit (QIAGEN)

Materials

• Bacterial overnight liquid cultures (1 - 5 mL)

• QIAprep Spin Miniprep Kit

Procedure

• Pellet 1 -5 mL bacterial culture by centrifugation at more than 8000 rpm for 3 minutes

• Resuspend pelleted bacterial cells in 250 μL P1 buffer and transfer to a microcentrifuge tube

• Add 250 μL P2 buffer and mix by inverting tube 4 – 6 times

• Add 350 μL N3 buffer and mix by inverting tube 4- 6 times

• Centrifuge for 10 min at 13000 rpm

• Apply supernatant to the QIAprep spin column by pipetting, centrifuge for 30 – 60 seconds and discard flow-through

• Wash the QIAprep spin column by adding 0.5 mL PB buffer, centrifuge for 30 – 60 seconds and discard flow-through

• Wash the QIAprep spin column by adding 0.75 mL PE buffer, centrifuge for 30 – 60 seconds and discard flow-through

• Centrifuge for 1 minute to remove residual wash buffer

• Elute DNA by placing QIAprep column in a clean 1.5 mL microcentrifuge tube and adding 30 or 50 μL EB buffer or water. Let stand for 1 minute and centrifuge for 1 minute

Phusion PCR

Materials

• 5X Phusion HF or GC Buffer

• dNTPs

• Forward and Reverse Primers

• Template plasmid DNA

• Phusion DNA polymerase

• Nuclease Free Water

Procedure

• Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:

Component 20 μL reaction 50 μL reaction
5X Phusion HF or GC Buffer 4 μL 10 μL
10 mM dNTPs 0.4 μL 1 μL
10 mM Forward Primer 1 μL 2.5 μL
10 mM Reverse Primer 1 μL 2.5 μL
Template plasmid DNA 1 pg – 10 ng 1 pg – 10 ng
Phusion DNA Polymerase 0.2 μL 0.5 μL
Nuclease Free Water to 20 μL to 50 μL

Usually 100 pg – 1 ng of template DNA is sufficient

• Mix by pipetting up and down or flicking the reactions

• Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:

Step Temperature Time
Initial Denaturation 98°C 30 seconds
25 – 35 cycles Denaturation 98°C 5 - 10 seconds
Annealing 45 – 72°C 10 – 30 seconds
Extension 72°C 15 -30 seconds per kb
Final Extension 72°C 5 -10 minutes
Hold 4°C

Guidelines

To be completed

Taq PCR

Materials

• 10X Standard Taq Reaction Buffer

• dNTPs

• Forward and Reverse Primers

• Template plasmid DNA

• Taq DNA polymerase

• Nuclease Free Water

Procedure

• Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:

Component 25 μL reaction 50 μL reaction
10X Standard Taq Reaction Buffer 2.5 μL 5 μL
10 mM dNTPs 0.5 μL 1 μL
10 mM Forward Primer 0.5 μL 1 μL
10 mM Reverse Primer 0.5 μL 1 μL
Template plasmid DNA 1 pg – 1 ng 1 pg – 1 ng
Taq DNA Polymerase 0.125 μL 0.25 μL
Nuclease Free Water to 25 μL to 50 μL

Usually 100 pg – 1 ng of template DNA is sufficient

• Mix by pipetting up and down or flicking the reactions

• Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:

Step Temperature Time
Initial Denaturation 95°C 30 seconds
25 – 35 cycles Denaturation 95°C 15 – 30 seconds
Annealing 45 – 68°C 15 – 60 seconds
Extension 68°C 1 minutes per kb
Final Extension 68°C 5 minutes
Hold 4°C

Guidelines

To be completed

Still under construction

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