Difference between revisions of "Team:KU Leuven/Research/Methods"
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<dd>1. Make an overnight culture of <i>E. coli</i> MG1655. </dd> | <dd>1. Make an overnight culture of <i>E. coli</i> MG1655. </dd> | ||
− | <dd>2. Take 500 | + | <dd>2. Take 500 µL overnight culture and add the phage P1. Incubate overnight at 37°C. </dd> |
− | <dd>3. Take single plaques of the P1 stock plate and bring this in a sterile | + | <dd>3. Take single plaques of the P1 stock plate and bring this in a sterile Eppendorf tube together with 200 µL of mQ.</dd> |
<dd>4. Overnight extraction while shaking at 37°C.</dd> | <dd>4. Overnight extraction while shaking at 37°C.</dd> | ||
− | <dd>5. Add 0.01, 0.1, 10 and 100 | + | <dd>5. Add 0.01, 0.1, 10 and 100 µL of extraction to 500 µL of a stationary phase culture of <i>E. coli</i> MG1655. Vortex and plate out.</dd> |
<dd>6. Add LB soft agar containing 10 mM MgSO<sub>4</sub> and 5 mM CaCl<sub>2</sub> and incubate at 37°C.</dd> | <dd>6. Add LB soft agar containing 10 mM MgSO<sub>4</sub> and 5 mM CaCl<sub>2</sub> and incubate at 37°C.</dd> | ||
− | <dd>7. | + | <dd>7. Choose the plate with the best lysates.</dd> |
− | <dd>8. Sterilize your spoon | + | <dd>8. Sterilize your spoon using a Bunsen burner, cool it down with water and wash it with 100% ethanol.</dd> |
− | <dd>9. Cut the soft agar and put this in a | + | <dd>9. Cut out a plaque from the soft agar and put this in a 10 mL syringe.</dd> |
− | <dd>10. Press the content of the syringe in an | + | <dd>10. Press the content of the syringe in an Eppendorf tube and centrifuge for 10 minutes at 14000 rpm.</dd> |
− | <dd>11. Take 650 µl and bring this in a new | + | <dd>11. Take 650 µl and bring this in a new Eppendorf tube.</dd> |
− | <dd>12. Extraction with 30 | + | <dd>12. Extraction with 30 µL of CHCl<sub>3</sub> </dd> |
− | <dd>13.Vortex | + | <dd>13.Vortex vigorously</dd> |
<dd>14. Store lysate at 4°C.</dd> | <dd>14. Store lysate at 4°C.</dd> | ||
− | <dt> 2. Preparation of lysate of donor strain.</dt> | + | <dt> 2. Preparation of the lysate of donor strain.</dt> |
− | <dd>1. | + | <dd>1. Firstly, centrifuge the lysate to ensure the chloroform is at the bottom of the Eppendorf tube. Then add 0.1, 1, 10 and 100 µl of lysate to 500 µL stationary phase overnight culture of the donor strain.</dd> |
− | <dd>2. Add LB soft agar containing 10 mM MgSO<sub>4</sub> and 5 mM CaCl<sub>2</sub>. Incubate this at 37°C. </dd> | + | <dd>2. Add LB soft agar containing 10 mM MgSO<sub>4</sub> and 5 mM CaCl<sub>2</sub>. Incubate this at 37°C.</dd> |
− | <dd>3. Sterilize your spoon in a | + | <dd>3. Sterilize your spoon in a Bunsen flame, cool it down with water and wash with 100% ethanol.</dd> |
− | <dd>4. Centrifuge the | + | <dd>4. Centrifuge the Eppendorf tubes 10 minutes at 14000 rpm.</dd> |
− | <dd>5. | + | <dd>5. Transfer 650 µL into a new Eppendorf tube </dd> |
− | <dd>6. | + | <dd>6. Extract with 30 µL of CHCl<sub>3</sub> </dd> |
− | <dd>7. Vortex | + | <dd>7. Vortex vigorously </dd> |
<dd>8. Store the lysate at 4°C.</dd> | <dd>8. Store the lysate at 4°C.</dd> | ||
</dt> | </dt> | ||
<dt> 3. Transduction to acceptor strain.</dt> | <dt> 3. Transduction to acceptor strain.</dt> | ||
− | <dd>1. Concentrate 500 | + | <dd>1. Concentrate 500 µL of stationary phase overnight acceptor strain culture five times in LB with 10 mM MgSO<sub>4</sub> and 5 mM CaCl<sub>2</sub> </dd> |
− | <dd>2. Add 0.1, 1, 10 and 100 | + | <dd>2. Add 0.1, 1, 10 and 100 µL of donor strain lysate to 100 µL acceptor strain. </dd> |
− | <dd>3. Incubate | + | <dd>3. Incubate 30 minutes at 37°C.</dd> |
− | <dd>4. Plate out on | + | <dd>4. Plate out on selective medium and incubate overnight. </dd> |
− | <dd>5. | + | <dd>5. Also plate out lysate to check for contamination.</dd> |
</dl> | </dl> | ||
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<p><b>Protocol :</b></p> | <p><b>Protocol :</b></p> | ||
<dl> | <dl> | ||
− | <dd>1. Transfer the liquid cultures to an | + | <dd>1. Transfer the liquid cultures to an Eppendorf tube and spin down a 13400 rpm for 1 à 2 minutes</dd> |
<dd>2. Resuspend the pelleted cels in 250 µL of the Resuspension solution. The cells should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.</dd> | <dd>2. Resuspend the pelleted cels in 250 µL of the Resuspension solution. The cells should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.</dd> | ||
<dd>3. Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. <br/> | <dd>3. Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. <br/> | ||
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<a href="http://www.genzyme.be/"><img src="https://static.igem.org/mediawiki/2015/b/b7/KU_Leuven_Genzyme_logo_transparant.png" alt="Genzyme" width="95%"></a> | <a href="http://www.genzyme.be/"><img src="https://static.igem.org/mediawiki/2015/b/b7/KU_Leuven_Genzyme_logo_transparant.png" alt="Genzyme" width="95%"></a> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="Eppendorf"> |
<a href="https://www.eppendorf.com/BE-en/"><img src="https://static.igem.org/mediawiki/2015/9/96/KU_Leuven_Logo_Eppendorf_transparant.png" alt="Eppendorf" width="95%"></a> | <a href="https://www.eppendorf.com/BE-en/"><img src="https://static.igem.org/mediawiki/2015/9/96/KU_Leuven_Logo_Eppendorf_transparant.png" alt="Eppendorf" width="95%"></a> | ||
</div> | </div> |
Revision as of 13:07, 16 September 2015
Methods
On this page you can find all of the methods and protocols used in the lab to obtain our results. For some techniques, we included some basic theory, since it is a prerequisite to get acquainted with the theory behind these techniques before using them. To learn more about them, click the titles below!
Contact
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be