Team:Bordeaux/NotebookJune11

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<h1>6<sup>th</sup> June </h1>
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<h1>11<sup>th</sup> June </h1>
<div class="type-exp"> Molecular biology</div>
<div class="type-exp"> Molecular biology</div>
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<ul class="day-detail">
<ul class="day-detail">
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<li>We continue our experiments to have BL21 without plasmid.</li><br>
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<li>Preparation of vector pSB1C3  for ligation with our PCR products (VPGXG-20, VPGXG-40 and VPGXG-60) : Digestion, ligation and transformation </li><br>
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<li>Preparation of  LB + Amp and LB + Chlo plates.</li>
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<li>Making LB+Chlo and LB+Amp plates</li></br>
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<li>Making competent cells: day 2</li>
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<BR><BR>
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<br><br>
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<div class="type-exp"> Production </div><br>
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A<sub>600</sub> preculture VPGXG-20 = 2,460</br>
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A<sub>600</sub> preculture VPGXG-40 = 2,678</br>
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    -> Culture of  VPGXG-20 and VPGXG-40 at 37°C<br><br>
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T=0, A<sub>600</sub> VPGXG-20 = 0,139<br>
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<div class="type-exp">Production</div>
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    A<sub>600</sub> VPGXG-40 = 0,072<br>
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    -> IPTG induction when A600 = 0,8 and culture at 25°C
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<BR><BR>
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<div class="type-exp"> Purification </div>
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<div class="title1">Protocol :</div>
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<ul class="day-detail">
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<ul class="strain-title">
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<li>30mL preculture of LB + Yeast Extract 5g/L + glycerol 1g/L + Amp at 37°C overnight</li>
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<li><b>Collection of cell lysate</b></li><br>
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<ul class ="strain-title">
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<ol type="-">
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<li>VPGXG-20</li>
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<li>Pour ELP cultures into 1L plastic centrifuge bottles</li>
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<li>VPGXG-40</li></ul>
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<li>Centrifuge 15min at 3,000 rpm and 4°C</li>
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<li>30mL preculture of LB + Yeast Extract 5g/L + glucose 1g/L + Amp at 37°C overnight</li>
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<li>Discard supernatant into 4L flask and bleach before disposal in the sink</li>
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<ul class="strain-title"><li>VPGXG-60</li></ul>
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<li>Repeat the latter steps if desired to consolidate cultures</li>
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<li>Resuspend cell pellet in 20mL PBS</li>
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<li>Transfer cell solution to a 50mL Falcon tube for sonication</li>
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<br><br>
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<li>Lyse cells by sonication on ice (3min: 10s on / 20s off – let sit on ice then repeat for a total of 2 sonication cycles)</li>
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<li>Transfer lysate into clear centrifuge tubes</li>
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<div class="type-exp">Purification</div>
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</ol></br>
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<li><b>ELP purification by Inverse Transition Cycling (ITC)</b></li>
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<ol type="-">
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<li>Add 10% polyethylenimine (PEI) (2mL per 1L culture) and gently mix</li>
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<li>Centrifuge at 4°C and 14,000 rpm for 15min</li>
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<li>Decant supernatant into clean centrifuge tube and discard pellet</li>
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<li>Allow solution to warm, add NaCl  (not exceeding 3M) and gently mix
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      - The solution should turn turbid with the ELP transition</li>
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<li>Centrifuge immediately after the ELP transition at room temperature and 14,000 rpm for 15min (HOT SPIN 1)</li>
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<li>Discard supernatant and resuspend ELP pellet in cold PBS(2mL per 1L culture)</li>
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<li>Transfer solution to 1.5mL labeled micro-centrifuge tubes</li>
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<li>Put tubes on ice 30min</li>
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<li>Centrifuge at 4°C and 14,000rpm for 10min (COLD SPIN 1)</li>
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<li>Decant or pipette supernatant into clear micro-centrifuges tube and discard pellet</li>
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<li>Allow the solution to warm and if necessary add 5M NaCl solution until ELP transition occurs </li>
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<li>Centrifuge at 37°C and 14,000rpm for 10min (HOT SPIN X)</li>
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<li>Discard supernatant, add cold buffer, and resuspend pellet</li>
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<li>Centrifuge at 4°C and 14,000rpm for 10min (COLD SPIN X)</li>
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<li>Decant or pipette supernatant into clear centrifuge tube and discard pellet</li>
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<li>Perform 3-5 rounds of ITC purification</li>
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</ol></ul>
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<br>
<br>
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Today we stop protocol after resuspend cell pellet in 20mL PBS and store at -80°C.
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<ul class="day-detail">
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<li>End of purification protocol and store at 4°C.</li></ul>
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{{:Team:Bordeaux/Pied}}
{{:Team:Bordeaux/Pied}}

Revision as of 21:36, 17 October 2014

11th June

Molecular biology

  • Preparation of vector pSB1C3 for ligation with our PCR products (VPGXG-20, VPGXG-40 and VPGXG-60) : Digestion, ligation and transformation

  • Making LB+Chlo and LB+Amp plates

  • Making competent cells: day 2


Production

  • 30mL preculture of LB + Yeast Extract 5g/L + glycerol 1g/L + Amp at 37°C overnight
    • VPGXG-20
    • VPGXG-40
  • 30mL preculture of LB + Yeast Extract 5g/L + glucose 1g/L + Amp at 37°C overnight
    • VPGXG-60


Purification

  • End of purification protocol and store at 4°C.

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