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

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                 <li><a href="#gibsonassembly">Gibson Assembly</a></li>
 
                 <li><a href="#gibsonassembly">Gibson Assembly</a></li>
 
                 <li><a href="#miniprep">Miniprep</a></li>
 
                 <li><a href="#miniprep">Miniprep</a></li>
 +
                <li><a href="#pcr">Polymerase Chain Reaction (PCR)</a></li>
  
 
     <!-- ADD SECTIONS HERE -->
 
     <!-- ADD SECTIONS HERE -->
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<h1>Miniprep - with QIAprep Spin Miniprep Kit (QIAGEN)</h1>
 
<h1>Miniprep - with QIAprep Spin Miniprep Kit (QIAGEN)</h1>
 
<h2>Materials</h2>
 
<h2>Materials</h2>
−
<p> • Overnight liquid cell cultures (5 – 8 mL)
+
<p> • Bacterial overnight liquid cultures (1 - 5 mL)
 
<p> • QIAprep Spin Miniprep Kit
 
<p> • QIAprep Spin Miniprep Kit
 
<h2>Procedure</h2>
 
<h2>Procedure</h2>
−
<p> • Centrifuge liquid cell cultures at 4000 rpm for 10 minutes</p>
+
<p> • Pellet 1 -5 mL bacterial culture by centrifugation at more than 8000 rpm for 3 minutes</p>
−
<p> to be continued, I'm going Paleo :) </p>
+
<p> • Resuspend pelleted bacterial cells in 250 μL P1 buffer and transfer to a microcentrifuge tube</p>
 +
<p> •  Add 250 μL P2 buffer and mix by inverting tube 4 – 6 times</p>
 +
<p> • Add 350 μL N3 buffer and mix by inverting tube 4- 6 times</p>
 +
<p> • Centrifuge for 10 min at 13000 rpm</p>
 +
<p> • Apply supernatant to the QIAprep spin column by pipetting, centrifuge for 30 – 60 seconds and discard flow-through</p>
 +
<p> • Wash the QIAprep spin column by adding 0.5 mL PB buffer, centrifuge for 30 – 60 seconds and discard flow-through</p>
 +
<p> • Wash the QIAprep spin column by adding 0.75 mL PE buffer, centrifuge for 30 – 60 seconds and discard flow-through</p>
 +
<p> • Centrifuge for 1 minute to remove residual wash buffer</p>
 +
<p> •  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</p>
 +
</div>
 +
 
 +
<!-- PCR -->
 +
<div id="pcr" class="well">       
 +
<h1>Polymerase Chain Reaction (PCR)</h1>
 +
<!-- PHUSION PCR -->
 +
<h1>Phusion PCR</h1>
 +
<h2>Materials</h2>
 +
<p> • 5X Phusion HF or GC Buffer</p>
 +
<p> • dNTPs</p>
 +
<p> • Forward and Reverse Primers</p>
 +
<p> • Template plasmid DNA</p>
 +
<p> • Phusion DNA polymerase</p>
 +
<p> • Nuclease Free Water</p>
 +
<h2>Procedure</h2>
 +
<p> • Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:</p>
 +
<table width="100%">
 +
<tr>
 +
<th>Component</th>
 +
<th>20 μL reaction</th>
 +
<th>50  μL reaction</th>
 +
</tr>
 +
<tr>
 +
<td>5X Phusion HF or GC Buffer</td>
 +
<td>4 μL</td>
 +
<td>10 μL</td>
 +
</tr>
 +
<tr>
 +
<td>10 mM dNTPs</td>
 +
<td>0.4 μL</td>
 +
<td>1 μL</td>
 +
</tr>
 +
<tr>
 +
<td>10 mM Forward Primer</td>
 +
<td>1 μL</td>
 +
<td>2.5 μL</td>
 +
</tr>
 +
<tr>
 +
<td>10 mM Reverse Primer</td>
 +
<td>1 μL</td>
 +
<td>2.5 μL</td>
 +
</tr>
 +
<tr>
 +
<td>Template plasmid DNA</td>
 +
<td>1 pg – 10 ng</td>
 +
<td>1 pg – 10 ng</td>
 +
</tr>
 +
<tr>
 +
<td>Phusion DNA Polymerase</td>
 +
<td>0.2 μL</td>
 +
<td>0.5 μL</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease Free Water</td>
 +
<td>to 20 μL</td>
 +
<td>to 50 μL</td>
 +
</tr>
 +
</table>
 +
          <p>        Usually 100 pg – 1 ng of template DNA is sufficient</p>
 +
<p> • Mix by pipetting up and down or flicking the reactions
 +
<p> • Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:</p>
 +
<table width="100%">
 +
<tr>
 +
<th>Step</th>
 +
<th>  </th>
 +
<th>Temperature</th>
 +
<th>Time</th>
 +
</tr>
 +
<tr>
 +
<td>Initial Denaturation</td>
 +
<td>  </td>
 +
<td>98°C</td>
 +
<td>30 seconds</td>
 +
</tr>
 +
<tr>
 +
<td>25 – 35 cycles</td>
 +
<td>Denaturation</td>
 +
<td>98°C</td>
 +
<td>5 - 10 seconds</td>
 +
</tr>
 +
<tr>
 +
<td>  </td>
 +
<td>Annealing</td>
 +
<td>45 – 72°C</td>
 +
<td>10 – 30 seconds</td>
 +
</tr>
 +
<tr>
 +
<td>  </td>
 +
<td>Extension</td>
 +
<td>72°C</td>
 +
<td>15 -30 seconds per kb</td>
 +
</tr>
 +
<tr>
 +
<td>Final Extension</td>
 +
<td>  </td>
 +
<td>72°C</td>
 +
<td>5 -10 minutes</td>
 +
</tr>
 +
<tr>
 +
<td>Hold</td>
 +
<td>  </td>
 +
<td>4°C </td>
 +
<td>  </td>
 +
</tr>
 +
</table>
 +
<h2>Guidelines</h2>
 +
<p>To be completed</p>
 +
<!-- TAQ PCR -->
 +
<h1>Taq PCR</h1>
 +
<h2>Materials</h2>
 +
<p> • 10X Standard Taq Reaction Buffer</p>
 +
<p> • dNTPs</p>
 +
<p> • Forward and Reverse Primers</p>
 +
<p> • Template plasmid DNA</p>
 +
<p> • Taq DNA polymerase</p>
 +
<p> • Nuclease Free Water</p>
 +
<h2>Procedure</h2>
 +
<p> • Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:</p>
 +
<table width="100%">
 +
<tr>
 +
<th>Component</th>
 +
<th>25 μL reaction</th>
 +
<th>50 μL reaction</th>
 +
</tr>
 +
<tr>
 +
<td>10X Standard Taq Reaction Buffer</td>
 +
<td>2.5 μL</td>
 +
<td>5 μL</td>
 +
</tr>
 +
<tr>
 +
<td>10 mM dNTPs</td>
 +
<td>0.5 μL</td>
 +
<td>1 μL</td>
 +
</tr>
 +
<tr>
 +
<td>10 mM Forward Primer</td>
 +
<td>0.5 μL</td>
 +
<td>1 μL</td>
 +
</tr>
 +
<tr>
 +
<td>10 mM Reverse Primer</td>
 +
<td>0.5 μL</td>
 +
<td>1 μL</td>
 +
</tr>
 +
<tr>
 +
<td>Template plasmid DNA</td>
 +
<td>1 pg – 1 ng</td>
 +
<td>1 pg – 1 ng</td>
 +
</tr>
 +
<tr>
 +
<td>Taq DNA Polymerase</td>
 +
<td>0.125 μL</td>
 +
<td>0.25 μL</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease Free Water</td>
 +
<td>to 25 μL</td>
 +
<td>to 50 μL</td>
 +
</tr>
 +
</table>
 +
            <p>        Usually 100 pg – 1 ng of template DNA is sufficient</p>
 +
<p> • Mix by pipetting up and down or flicking the reactions
 +
<p> • Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:</p>
 +
<table width="100%">
 +
<tr>
 +
<th>Step</th>
 +
<th>  </th>
 +
<th>Temperature</th>
 +
<th>Time</th>
 +
</tr>
 +
<tr>
 +
<td>Initial Denaturation</td>
 +
<td>  </td>
 +
<td>95°C</td>
 +
<td>30 seconds</td>
 +
</tr>
 +
<tr>
 +
<td>25 – 35 cycles</td>
 +
<td>Denaturation</td>
 +
<td>95°C</td>
 +
<td>15 – 30 seconds</td>
 +
</tr>
 +
<tr>
 +
<td>  </td>
 +
<td>Annealing</td>
 +
<td>45 – 68°C</td>
 +
<td>15 – 60 seconds</td>
 +
</tr>
 +
<tr>
 +
<td>  </td>
 +
<td>Extension</td>
 +
<td>68°C</td>
 +
<td>1 minutes per kb</td>
 +
</tr>
 +
<tr>
 +
<td>Final Extension</td>
 +
<td>  </td>
 +
<td>68°C</td>
 +
<td>5 minutes</td>
 +
</tr>
 +
<tr>
 +
<td>Hold</td>
 +
<td>  </td>
 +
<td>4°C </td>
 +
<td>  </td>
 +
</tr>
 +
</table>
 +
<h2>Guidelines</h2>
 +
<p>To be completed</p>
 +
</div>
  
 
     <!-- SECTION 4 -->
 
     <!-- SECTION 4 -->
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         </div>
 
         </div>
 
     </div>
 
     </div>
−
     <!-- Footer -->
+
 
 +
 
 +
     <!-- FOOTER -->
 
     <footer class="footer-distributed">
 
     <footer class="footer-distributed">
  

Revision as of 09:32, 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 - based on NEB Taq PCR Protocol

Materials

• 10X Standard Taq Reaction Buffer

• dNTPs

• Taq polymerase

• Forward and Reverse Primers

• Nuclease Free Water

• Petri dish with transformed colonies

Procedure

• Prepare following reaction in 0.5 mL PCR tubes on ice by making a Master Mix for all reactions and adding polymerase last:

Component 25 μL reaction Final concentration
10X Standard Taq Reaction Buffer 2.5 μL 1X
10 mM dNTPs 0.5 μL 200 μM
10 mM Forward Primer 0.5 μL 0.2 μM
10 mM Reverse Primer 0.5 μL 0.2 μM
Taq DNA Polymerase 0.125 μL 0.625 units/25 μL PCR
Nuclease Free Water to 25 μL

• 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 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 - 60seconds
Extension 68°C 1 minutes per kb
Final Extension 68°C 5 minutes
Hold 4°C

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

Polymerase Chain Reaction (PCR)

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

Section 4-1

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Section 4-2

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Still under construction

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