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− | <div class="summarytext1">
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− | <ul style = "list-style-type: none">
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− | <li><h4><a onclick="ShowHide('HiddenDiv')">P1 transduction</a></h4>
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− | <div class="mid" id="HiddenDiv" style="display: none"> <p style="text-align:left">
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− | </br>
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− | <ul>
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− | <li style = "text-align: left; list-style-type: none;" > 1. Preparation of lysate starting from stock plate of phage
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− | <ul>
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− | <li> <p2> Stockplate of E. coli MG1655 </p2> </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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− | </br>
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− | <li style = "text-align: left"> 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>
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− | <li>- Add LB softagar with 10 mM MgSO4 and 5 mM CaCl2. Incubate at 37°C.</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>- 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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− | </br>
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− | <li style = "text-align: left"> 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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− |
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− | </ul>
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− | </li>
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− | </ul>
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− | </p>
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− | </br>
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− | </div>
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− | </li>
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− | </br>
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− | <li>
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− | <h4><a onclick="ShowHide('HiddenDiv2')">Gibson Assembly</a></h4>
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− | <div class="mid" id="HiddenDiv2" style="display: none;"> <p>Het volledige protocol van 5</p></div></li>
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− | </br>
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− | <li><h4><a onclick="ShowHide('HiddenDiv3')">Miniprep</a></h4>
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− | <div class="mid" id="HiddenDiv3" style="display: none;"> <p>Het volledige protocol van 5</p></div></li>
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− | </br>
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− | <li><h4><a onclick="ShowHide('HiddenDiv4')">Gel purification</a></h4>
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− | <div class="mid" id="HiddenDiv4" style="display: none;"> <p>Het volledige protocol van 5</p></div></li>
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− | </br>
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− | <li><h4><a onclick="ShowHide('HiddenDiv5')">Chemocompetent cells</a></h4>
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− | <div class="mid" id="HiddenDiv5" style="display: none;">
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− | </br>
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− | <p style = "margin : 17px">
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− | Materials</p>
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− | <ul>
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− | <li> Single colony of E. coli cells to be transformed </li>
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− | <li> LB medium </li>
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− | <li> 0.1 M CaCl2, ice cold <li>
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− | <li> LB amp plates </li>
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− | <li> 42 °C water bath </li>
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− | <li> 0.1 M CaCl2+15% glycerol, sterile </li>
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− |
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− | <p> Procedure: </p>
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− | <ul>
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− | <li> 1. Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C overnight. </li>
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− | <li> 2. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask). Shake vigorously at 37 °C to OD600 ~ 0.25-0.3 (usually it takes about 1.5-hours). <li>
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− | <li>3. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice </li>
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− | <li>4. Centrifuge the cells for 10 min at 3300 g (e.g. 4,000 rpm in the Jouan tabletop centrifuge) at 4 °C. </li>
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− | <li>5. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. </li>
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− | <li>6. Keep the cells on ice for 30 min.</li>
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− | <li>7. Centrifuge the cells as above.</li>
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− | <li>8. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution plus 15% glycerol. </li>
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− | <li>9. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. </li>
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− | </br>
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− | <li> Freeze these tubes on dry ice and then transfer them to -70 C freezer. </li>
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− | </ul>
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− | </br>
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− | </div>
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− | </li>
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− | </br>
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− | <li><h4><a onclick="ShowHide('HiddenDiv6')">Electrocompetent cells</a></h4>
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− | <div class="mid" id="HiddenDiv6" style="display: none;">
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− | </br>
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− | <p style = "margin : 17px"> Materials: </p>
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− | <p>
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− | <ul>
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− | <li style = "text-align: left; list-style-type: none;"> 1L sterile LB without NaCl (10g tryptone, 5g yeast extract per 1L) </li>
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− | <li>500 mL of 10% v/v glycerol</li>
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− | <li>cold falcon tubes of 50 mL </li>
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− | <li>cold eppies and pipette tips </li>
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− | </ul>
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− | <p style = "margin : 17px;" > Procedure </p>
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− | <ul>
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− | <li style = "text-align: left; list-style-type: none;"> Day 1:
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− | <ul> <li> Strike your cells on a plate and grow overnight in 37°C. </li> </ul>
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− | </li>
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− | <li>Day 2:
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− | <ul> <li> Pick a single colony from your plate and grow it in 1-3 mL salt free LB overnight in 37°C. </li> </ul>
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− | </li>
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− | <li> Day 3:
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− | <ul>
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− | <li> 1. Grow 300-400 mL cells (without salt) in 37°C untill the OD reaches 0.6 (use a starting </li>
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− | culture).
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− | <li> 2. Cool down on ice and from now on perform all the steps in 4°C.</li>
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− | <li> 3. Spin the cells down in falcon tubes (3500 g, 20 min, 4°C). Using falcon tubes ensures no detergents present. </li>
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− | <li> 4. Resuspend the pellet in 30 mL icecold 10% glycerol (filtered to a disposable bottle to
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− | ensure no detergents). Spin down the cells (5000 g, 10 min, 4°C). Repeat this step 3 times. </li>
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− | <li> 5. Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not much more than 1.5 mL). </li>
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− | <li> 6. Take 50 µL sample and do the electroporation test (without DNA). You should have a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL glycerol. </li>
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− | <li> 7. Aliquot the cells (50 µL) and quick-freeze in liquid nitrogen and store at -80°C.</li>
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− | </li>
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− | </ul>
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− | </p>
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− | </br>
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− | </br>
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− | </div></li>
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− | </br>
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− |
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− | <li><h4><a onclick="ShowHide('HiddenDiv7')">Transformation</a></h4>
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− | <div class="mid" id="HiddenDiv7" style="display: none;"> <p>Het volledige protocol van 5</p></div></li>
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− | </ul>
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| <div class="summarytext1"> | | <div class="summarytext1"> |