Difference between revisions of "Team:KU Leuven/Research/Methods"

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<li><h4><a onclick="ShowHide('HiddenDiv')">P1 transduction</a></h4>
 
<li><h4><a onclick="ShowHide('HiddenDiv')">P1 transduction</a></h4>
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<div class="mid" id="HiddenDiv" style="display: none;"> <p>Het volledige protocol <br>
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<div class="mid" id="HiddenDiv" style="display: none;"> <p>
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Lorem Ipsum is slechts een proeftekst uit het drukkerij- en zetterijwezen. Lorem Ipsum is de standaard proeftekst in deze bedrijfstak sinds de 16e eeuw, toen een onbekende drukker een zethaak met letters nam en ze door elkaar husselde om een font-catalogus te maken. Het heeft niet alleen vijf eeuwen overleefd maar is ook, vrijwel onveranderd, overgenomen in elektronische letterzetting. Het is in de jaren '60 populair geworden met de introductie van Letraset vellen met Lorem Ipsum passages en meer recentelijk door desktop publishing software zoals Aldus PageMaker die versies van Lorem Ipsum bevatten.</p></div></li>
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<ul><li> 1. Preparation of lysate starting from stock plate of phage
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<li>- Stockplate of E. coli MG1655</li>
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<li>- O/N culture of E. coli MG1655</li>
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<li>- Take 500 µl of O/N culture and add P1 (-80°C). Incubate O/N at 37°C</li>
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<li>- Take single plaques of P1 stock plate and bring it in a sterile eppendorf tube together with
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200 µl of MQ</li>
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<li>- O/N extraction, shaking at 37°C</li>
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<li>- Add 0.01, 0.1, 1, 10 and 100 µl of extraction to 500 µl of a stationary phase culture of E. coli
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MG1655. Vortex and bring to a sterile petri dish with LB.</li>
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<li>- Add LB soft agar with 10 mM MgSO4 and 5 mM CaCl2 in it and incubate at 37°C.</li>
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<li>- Take plate with best lysis.</li>
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<li>- Sterilize spoon in bunsen flame, cool down with water and wash with 100% of ethanol.</li>
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<li>- Put soft agar in syringe (10 ml and G22).</li>
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<li>- Centrifugation of eppendorf tubes at maximal speed (14 000 rpm) during 10 minutes.</li>
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<li>- Take 650 µl and bring in new eppendorf tube (total 1300 µl).</li>
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<li>- Extraction with 30 µl of CHCl3</li>
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<li>- Vortex heavy!!!</li>
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<li>- Storage of lysate at 4°C. </li>
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</ul>
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</li>
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< li> 2. Preparation of lysate of donor strain.
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 +
<ul>
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<li>- Add to 500 µl aliquots of O/N stationary phase culture of donor strain 0.1, 1, 10 and 100 µl of
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 +
lysate. First, you have to centrifugate the lysate to be sure the chloroform is at the bottom of
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 +
the eppendorf tube.</li>
 +
 
 +
<li>- Add LB softagar with 10 mM MgSO4 and 5 mM CaCl2. Incubate at 37°C.</li>
 +
 
 +
<li>- Sterilize spoon in bunsen flame, cool down with water and wash with 100% of ethanol. </li>
 +
 
 +
<li>- Centrifugation of eppendorf tubes at maximal speed (14 000 rpm) during 10 minutes. </li>
 +
 
 +
<li>- Take 650 µl and bring in new eppendorf tube (total 1300 µl).</li>
 +
 
 +
<li>- Extraction with 30 µl of CHCl3</li>
 +
 
 +
<li>- Vortex heavy!!!</li>
 +
 
 +
<li>- Storage of lysate at 4°C.</li>
 +
</ul>
 +
</li>
 +
 
 +
<li> 3. Transduction to acceptor strain.
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<ul>
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<li> - Concentrate 500 µl of O/N stationary phase culture of acceptor strain 5 times in LB with 10 mM MgSO4 and 5 mM CaCl2. </li>
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 +
<li> - Add 0.1, 1, 10 and 100 µl of the donor strain lysate to 100 µl aliquots of the acceptor strain. </li>
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 +
<li> - Incubate 30 minutes at 37°C </li>
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<li> - Plate out on selective medium en incubate overnight </li>
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<li> -Plate also lysate out on normal LB plate to see if lysate contains no contamination.</li>
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</ul>
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</li>
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</ul>
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</p></div></li>
 
</br>
 
</br>
 
<li>
 
<li>

Revision as of 16:28, 1 September 2015

Logo


Methods

  • P1 transduction

    • < li> 2. Preparation of lysate of donor strain.
    • - Add to 500 µl aliquots of O/N stationary phase culture of donor strain 0.1, 1, 10 and 100 µl of lysate. First, you have to centrifugate the lysate to be sure the chloroform is at the bottom of the eppendorf tube.
    • - Add LB softagar with 10 mM MgSO4 and 5 mM CaCl2. Incubate at 37°C.
    • - Sterilize spoon in bunsen flame, cool down with water and wash with 100% of ethanol.
    • - Centrifugation of eppendorf tubes at maximal speed (14 000 rpm) during 10 minutes.
    • - Take 650 µl and bring in new eppendorf tube (total 1300 µl).
    • - Extraction with 30 µl of CHCl3
    • - Vortex heavy!!!
    • - Storage of lysate at 4°C.
  • 3. Transduction to acceptor strain.
    • - Concentrate 500 µl of O/N stationary phase culture of acceptor strain 5 times in LB with 10 mM MgSO4 and 5 mM CaCl2.
    • - Add 0.1, 1, 10 and 100 µl of the donor strain lysate to 100 µl aliquots of the acceptor strain.
    • - Incubate 30 minutes at 37°C
    • - Plate out on selective medium en incubate overnight
    • -Plate also lysate out on normal LB plate to see if lysate contains no contamination.


  • Gibson Assembly


  • Miniprep


  • Gel purification


  • Chemocompetent cells


  • Electrocompetent cells


  • Transformation