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

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<li><a href="#pcrpurification">PCR Product Purification</a></li>
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<li><a href="#pegliac">PEG/LiAc Solution</a></li>
  
 
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<p> • Wash the QIAprep spin column by adding 0.75 mL PE 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> • 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>
+
<p> •  Elute DNA by placing QIAprep column in a clean 1.5 mL microcentrifuge tube and adding 50 μL EB buffer or water (or less for higher concentration). Let stand for 1 minute and centrifuge for 1 minute</p>
 
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  <!-- PCR PURIFICATION -->
          
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<div id="pcrpurification" class="well">          
 +
<h1>PCR Product Purification - with QIAquick PCR Purification Kit (QIAGEN)</h1>
 +
<h2>Materials</h2>
 +
<p> • PCR products</p>
 +
<p> • QIAquick PCR Purification Kit</p>
 +
<h2>Procedure</h2>
 +
<p> • Add 5 volumes PB buffer to 1 volume of PCR product and mix</p>
 +
<p> • Place QIAquick column in 2 ml collection tube</p>
 +
<p> • Apply samples to QIAquick column and centrifuge for 30 – 60 seconds, discard flow-through</p>
 +
<p> • Wash by adding 0.75 μL PE buffer to QIAquick column and centrifuge fo 30 – 60 seconds, discard flow-through</p>
 +
<p> • Centrifuge QIAquick column for 1 minutes to remove residual wash buffer</p>
 +
<p> • Elute DNA by adding 30 or 50 μL EB buffer or water to the center of the QIAquick column. Let stand for 1 minutes and centrifuge for 1 minute</p>
 +
</div>
 +
 
 +
<!-- PEG LIAC SOLUTION -->
 +
<div id="pegliac" class="well">       
 +
<h1>PEG/LiAc Solution</h1>
 +
<h2>Materials</h2>
 +
<p> • 50% PEG  (Polyethylene glycol) prepared with sterile deionized water</p>
 +
<p> • 10X TE buffer: 0.1 M Tris-Hcl, 10 mM EDTA, ph 7.5, autoclaved</p>
 +
<p> • 10X LiAc: 1 M lithium acetate, pH 7.5 adjusted with dilute acetic acid, autoclaved</p>
 +
<h2>Procedure</h2>
 +
<p> • Prepare PEG/LiAc solution as follows:</p>
 +
<table width="100%">
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<tr>
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<th>Stock concentration</th>
 +
<th>Final concentration</th>
 +
<th>Total quantity for 10 mL solution</th>
 +
</tr>
 +
<tr>
 +
<td>50% PEG</td>
 +
<td>40% PEG</td>
 +
<td>8 mL</td>
 +
</tr>
 +
<tr>
 +
<td>10X TE buffer</td>
 +
<td>1X TE buffer</td>
 +
<td>1 mL</td>
 +
</tr>
 +
<tr>
 +
<td>10X LiAc</td>
 +
<td>1X LiAc</td>
 +
<td>1 mL</td>
 +
</tr>
 +
</table>
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 +
 
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             </div>
 
             </div>
 
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Revision as of 10:22, 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 (except template plasmid DNA)

• Petri dish with transformed colonies

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 tubes

• 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 50 μL EB buffer or water (or less for higher concentration). Let stand for 1 minute and centrifuge for 1 minute

Phusion PCR – based on NEB Phusion PCR Protocol

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 – based on NEB Taq PCR Protocol

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

PCR Product Purification - with QIAquick PCR Purification Kit (QIAGEN)

Materials

• PCR products

• QIAquick PCR Purification Kit

Procedure

• Add 5 volumes PB buffer to 1 volume of PCR product and mix

• Place QIAquick column in 2 ml collection tube

• Apply samples to QIAquick column and centrifuge for 30 – 60 seconds, discard flow-through

• Wash by adding 0.75 μL PE buffer to QIAquick column and centrifuge fo 30 – 60 seconds, discard flow-through

• Centrifuge QIAquick column for 1 minutes to remove residual wash buffer

• Elute DNA by adding 30 or 50 μL EB buffer or water to the center of the QIAquick column. Let stand for 1 minutes and centrifuge for 1 minute

PEG/LiAc Solution

Materials

• 50% PEG (Polyethylene glycol) prepared with sterile deionized water

• 10X TE buffer: 0.1 M Tris-Hcl, 10 mM EDTA, ph 7.5, autoclaved

• 10X LiAc: 1 M lithium acetate, pH 7.5 adjusted with dilute acetic acid, autoclaved

Procedure

• Prepare PEG/LiAc solution as follows:

Stock concentration Final concentration Total quantity for 10 mL solution
50% PEG 40% PEG 8 mL
10X TE buffer 1X TE buffer 1 mL
10X LiAc 1X LiAc 1 mL

Still under construction