Difference between revisions of "Team:Paris Bettencourt/Protocols"

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<table style="width=35%">
 +
  <tr>
 +
    <td><b>Electroporation Protocol</b><br>
 +
    <ul>
 +
      <li>Thaw electrocompetent cells on ice
 +
      <li>Add 2µL of ligation product or 0.5µL of native plasmid to the cells
 +
      <li>Transfer the cells in an 0.2mm electroporation cuvette
 +
      <li>put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF
 +
      <li>add 200µL of LB right after pulsing
 +
      <li>recover 2 hours at 37°C
 +
      <li>plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C
 +
    </ul>
 +
    </td>
 +
  </tr>
 +
</table>
 +
 
 +
 
 +
<table style="width=30%">
 +
  <tr>
 +
    <td><b>Analytical digestion protocol</b><br>
 +
    <ul>
 +
      <li>Prepare the following mix:
 +
      <ul>
 +
        <li>2µL 10X Digestion buffer
 +
        <li>0.5µL Eco31I
 +
        <li>0.5µL BbsI
 +
        <li>2µL of DNA (200ng)
 +
        <li>15µL water
 +
      </ul>
 +
      <li>incube 1h at 37°C
 +
    </ul>
 +
    </td>
 +
  </tr>
 +
</table>
 +
 
 +
 
 +
<table style="width=35%">
 +
  <tr>
 +
    <td><b>Electrocompetent Cells Preparation Protocol</b>
 +
    <ul>
 +
      <li>Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
 +
      <li>incubate until the the DO600 reach 0.5 to 0.7
 +
      <li>place the cultures on ice for 15 minutes
 +
      <li>pour the culture in cold sterile 50mL falcon tubes
 +
      <li>centrifuge them for 10 minutes at 6000rpm
 +
      <li>throw the supernatant
 +
      <li>resuspend the cells in 50mL cold distilled water
 +
      <li>centrifuge them for 10 minutes at 6000rpm
 +
      <li>throw the supernatant
 +
      <li>resuspend the cells in 25mL cold distilled water
 +
      <li>centrifuge them for 10 minutes at 6000rpm
 +
      <li>throw the supernatant
 +
      <li>resuspend the cells in 12.5mL cold 10% glycerol
 +
      <li>centrifuge them for 10 minutes at 6000rpm
 +
      <li>throw the supernatant
 +
      <li>resuspend the cells in 5mL cold 10% glycerol
 +
      <li>make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C
 +
    </ul>
 +
    </td>
 +
  </tr>
 +
</table>
 +
 
 +
 
 +
<table style="width=%">
 +
  <tr>
 +
    <td><b>Heat Shock transformation protocol</b><br/>
 +
    <ul>
 +
      <li>Thaw frozen chemically competent cells (20µL aliquots) on ice for 10min.
 +
      <li>add 2µL of ligation product (or 0.5µL of miniprep) and incubate the cells 30sec at 42°C.
 +
      <li>put the cells back on ice for 2min.
 +
      <li>add 200µL of LB to the cells and incubate 2 hours at 37°C.
 +
      <li>plate the cells on LB + erythromycin (150µg/mL) or LB + erythromycin (10µg/mL), incubation at 37°C overnight.
 +
    </ul>
 +
    </td>
 +
  </tr>
 +
</table>
 +
 
 +
 
 +
<table style="30%">
 +
  <tr>
 +
    <td><b>Ligation Protocol</b><br>
 +
    <ul>
 +
      <li>Mix the following
 +
 
 +
      <ul>
 +
        <li>vector 100ng
 +
        <li>insert 300ng
 +
        <li>T4 DNA ligase buffer 10X
 +
        <li>T4 DNA ligase
 +
        <li>up to 20µL of water
 +
      </ul>
 +
      <li>incubate 30 to 40 minutes at room temperature
 +
    </ul>
 +
    </td>
 +
  </tr>
 +
</table>
 +
 
 +
<table  style="width:35%">
 +
  <tr>
 +
    <td><b>Digestion Protocol</b><br/>
 +
<ul>
 +
<li>Prepare the following mix:<br/>
 +
  <ul>
 +
  <li>4μL of Enzyme 1
 +
  <li>4μL of Enzyme 2
 +
  <li>4μL of FastAP
 +
  <li>12μL of Fast Digest buffer 10X
 +
  <li>1 to 3 μg of DNA
 +
  <li>up to 120μL of water
 +
  </ul>
 +
<li>mix by pipetting up and down
 +
<li>incube 10min at 37°C
 +
</ul>
 +
    </td>
 +
  </tr>
 +
 
 +
</table>
 +
 
 +
 
 +
<table style="width:75%">
 +
  <tr>
 +
    <td><b>Miniprep protocol using a QIAGEN kit </b>
 +
<ul>
 +
<li>centrifuge an overnight culture of cells 10min at 4krpm
 +
<li>throw the filtrate
 +
<li>resuspend the pellet in 250μL of Cell Resuspension Solution then mix it
 +
<li>transfer it in a 1.5mL microcentrifuge tube
 +
<li>add 250mL of Cell lysis solution and mix by inverting several times
 +
<li>incube until the liquid is clear, maximum 5min
 +
<li>add 10μL of Alkalyne protease solution, mix by inverting several times and incube 3 to 4 min
 +
<li>add 350μL of Neutralisation Solution and mix by inverting several times
 +
<li>centrifuge 10min at 14krpm
 +
<li>add the supernatant in a column
 +
<li>centrifuge 1min, 14krpm, then throw the filtrat
 +
<li>add 750μL Washing Solution
 +
<li>centrifuge 1min, 14krpm, then throw the filtrat
 +
<li>add 250μL Washing Solution
 +
<li>centrifuge 2min, 14krpm, then throw the filtrat
 +
<li>centrifuge 1min, 14krpm
 +
<li>transfer the column in a sterile microcentrifuge 1.5ml tube
 +
<li>add 50μL of DNAse/RNAse free water right on the membrane of the filter, wait 1min
 +
<li>centrifuge 1min, 14krpm
 +
<li>throw the column, plasmid is saved in water
 +
</ul>
 +
    </td>
 +
  </tr>
 +
</table>
 +
 
 +
 
 +
<table>
 +
  <tr>
 +
    <td><b>Annealing Protocol</b>
 +
<ul>
 +
 
 +
<li>Phosphorylation of the oligos
 +
<ul>
 +
  <li>        5.6μL DNAse/RNAse free water
 +
  <li> 6.0μL  o15.011 (10µM)
 +
  <li> 6.0μL  o15.012 (10µM)
 +
  <li> 2.0μL  10X T4 DNA ligase buffer
 +
  <li> 0.4μL  T4 PolyNucleotide Kinase
 +
Total:  20μL
 +
 
 +
</ul>
 +
<br/>
 +
 
 +
  <li>incube 30min at 37°C
 +
  <li>add 1μL of 1M NaCl
 +
  <li>incube 5min at 95°C
 +
  <li>let the mix cool down
 +
  <li>use 2μL of the mix as a 10X solution
 +
</ul>
 +
    </tr>
 +
  </td>
 +
</table>
 +
 
 +
 
 +
<table style="width:50%">
 +
  <tr>
 +
    <td>
 +
<ul>
 +
<b>PCR purification protocol</b>
 +
<li>Add 5 volumes of resuspension buffer to 1 volume of PCR product in an 1.5mL microcentrifuge tube, mix by pipetting up and down
 +
<li>Transfer in a centrifugation column
 +
<li>Centrifuge 1 min at 14000 rpm
 +
<li>Throw the filtration product
 +
<li>Add 700μL of washing solution
 +
<li>Centrifuge 1 min at 14000 rpm
 +
<li>Throw the filtration product
 +
<li>Add 500μL of washing solution
 +
<li>Centrifuge 1 min at 14000 rpm
 +
<li>Throw the filtration product
 +
<li>Centrifuge 1 min at 14000 rpm
 +
<li>Throw the filtration product
 +
<li>Put the column in a sterile 1.5mL microcentrifuge tube
 +
<li>Add 45μL of DNAse/RNAse free water on the membrane
 +
<li>Wait 2 minutes
 +
<li>Centrifuge 2min at 10000rpm
 +
<li>Discard the column, DNA is saved in water
 +
    </td>
 +
  </tr>
 +
</table>

Revision as of 13:23, 12 August 2015

Electroporation Protocol
  • Thaw electrocompetent cells on ice
  • Add 2µL of ligation product or 0.5µL of native plasmid to the cells
  • Transfer the cells in an 0.2mm electroporation cuvette
  • put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF
  • add 200µL of LB right after pulsing
  • recover 2 hours at 37°C
  • plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C


Analytical digestion protocol
  • Prepare the following mix:
    • 2µL 10X Digestion buffer
    • 0.5µL Eco31I
    • 0.5µL BbsI
    • 2µL of DNA (200ng)
    • 15µL water
  • incube 1h at 37°C


Electrocompetent Cells Preparation Protocol
  • Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
  • incubate until the the DO600 reach 0.5 to 0.7
  • place the cultures on ice for 15 minutes
  • pour the culture in cold sterile 50mL falcon tubes
  • centrifuge them for 10 minutes at 6000rpm
  • throw the supernatant
  • resuspend the cells in 50mL cold distilled water
  • centrifuge them for 10 minutes at 6000rpm
  • throw the supernatant
  • resuspend the cells in 25mL cold distilled water
  • centrifuge them for 10 minutes at 6000rpm
  • throw the supernatant
  • resuspend the cells in 12.5mL cold 10% glycerol
  • centrifuge them for 10 minutes at 6000rpm
  • throw the supernatant
  • resuspend the cells in 5mL cold 10% glycerol
  • make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C


Heat Shock transformation protocol
  • Thaw frozen chemically competent cells (20µL aliquots) on ice for 10min.
  • add 2µL of ligation product (or 0.5µL of miniprep) and incubate the cells 30sec at 42°C.
  • put the cells back on ice for 2min.
  • add 200µL of LB to the cells and incubate 2 hours at 37°C.
  • plate the cells on LB + erythromycin (150µg/mL) or LB + erythromycin (10µg/mL), incubation at 37°C overnight.


Ligation Protocol
  • Mix the following
    • vector 100ng
    • insert 300ng
    • T4 DNA ligase buffer 10X
    • T4 DNA ligase
    • up to 20µL of water
  • incubate 30 to 40 minutes at room temperature
Digestion Protocol
  • Prepare the following mix:
    • 4μL of Enzyme 1
    • 4μL of Enzyme 2
    • 4μL of FastAP
    • 12μL of Fast Digest buffer 10X
    • 1 to 3 μg of DNA
    • up to 120μL of water
  • mix by pipetting up and down
  • incube 10min at 37°C


Miniprep protocol using a QIAGEN kit
  • centrifuge an overnight culture of cells 10min at 4krpm
  • throw the filtrate
  • resuspend the pellet in 250μL of Cell Resuspension Solution then mix it
  • transfer it in a 1.5mL microcentrifuge tube
  • add 250mL of Cell lysis solution and mix by inverting several times
  • incube until the liquid is clear, maximum 5min
  • add 10μL of Alkalyne protease solution, mix by inverting several times and incube 3 to 4 min
  • add 350μL of Neutralisation Solution and mix by inverting several times
  • centrifuge 10min at 14krpm
  • add the supernatant in a column
  • centrifuge 1min, 14krpm, then throw the filtrat
  • add 750μL Washing Solution
  • centrifuge 1min, 14krpm, then throw the filtrat
  • add 250μL Washing Solution
  • centrifuge 2min, 14krpm, then throw the filtrat
  • centrifuge 1min, 14krpm
  • transfer the column in a sterile microcentrifuge 1.5ml tube
  • add 50μL of DNAse/RNAse free water right on the membrane of the filter, wait 1min
  • centrifuge 1min, 14krpm
  • throw the column, plasmid is saved in water


</td>
Annealing Protocol
  • Phosphorylation of the oligos
    • 5.6μL DNAse/RNAse free water
    • 6.0μL o15.011 (10µM)
    • 6.0μL o15.012 (10µM)
    • 2.0μL 10X T4 DNA ligase buffer
    • 0.4μL T4 PolyNucleotide Kinase Total: 20μL


  • incube 30min at 37°C
  • add 1μL of 1M NaCl
  • incube 5min at 95°C
  • let the mix cool down
  • use 2μL of the mix as a 10X solution


    PCR purification protocol
  • Add 5 volumes of resuspension buffer to 1 volume of PCR product in an 1.5mL microcentrifuge tube, mix by pipetting up and down
  • Transfer in a centrifugation column
  • Centrifuge 1 min at 14000 rpm
  • Throw the filtration product
  • Add 700μL of washing solution
  • Centrifuge 1 min at 14000 rpm
  • Throw the filtration product
  • Add 500μL of washing solution
  • Centrifuge 1 min at 14000 rpm
  • Throw the filtration product
  • Centrifuge 1 min at 14000 rpm
  • Throw the filtration product
  • Put the column in a sterile 1.5mL microcentrifuge tube
  • Add 45μL of DNAse/RNAse free water on the membrane
  • Wait 2 minutes
  • Centrifuge 2min at 10000rpm
  • Discard the column, DNA is saved in water </td>