Difference between revisions of "Team:KU Leuven/Research/Methods"
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<dd>10. Press the content of the syringe in an Eppendorf tube and centrifuge for 10 minutes at 14000 rpm.</dd> | <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 Eppendorf tube.</dd> | <dd>11. Take 650 µl and bring this in a new Eppendorf tube.</dd> | ||
− | <dd>12. Extraction with 30 µL of CHCl<sub>3</sub> </dd> | + | <dd>12. Extraction with 30 µL of CHCl<sub>3</sub>. </dd> |
− | <dd>13.Vortex vigorously</dd> | + | <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 the lysate of donor strain.</dt> | <dt> 2. Preparation of the lysate of donor strain.</dt> | ||
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<dd>3. Sterilize your spoon in a Bunsen flame, cool it down with water and wash with 100% ethanol.</dd> | <dd>3. Sterilize your spoon in a Bunsen flame, cool it down with water and wash with 100% ethanol.</dd> | ||
<dd>4. Centrifuge the Eppendorf tubes 10 minutes at 14000 rpm.</dd> | <dd>4. Centrifuge the Eppendorf tubes 10 minutes at 14000 rpm.</dd> | ||
− | <dd>5. Transfer 650 µL into a new Eppendorf tube </dd> | + | <dd>5. Transfer 650 µL into a new Eppendorf tube. </dd> |
− | <dd>6. Extract with 30 µL of CHCl<sub>3</sub> </dd> | + | <dd>6. Extract with 30 µL of CHCl<sub>3</sub>. </dd> |
− | <dd>7. Vortex vigorously </dd> | + | <dd>7. Vortex vigorously. </dd> |
<dd>8. Store the lysate at 4°C.</dd> | <dd>8. Store the lysate at 4°C.</dd> | ||
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<dd>3. Incubate 30 minutes at 37°C.</dd> | <dd>3. Incubate 30 minutes at 37°C.</dd> | ||
<dd>4. Plate out on selective medium and incubate overnight. </dd> | <dd>4. Plate out on selective medium and incubate overnight. </dd> | ||
− | <dd>5. | + | <dd>5. Plate out lysate-only to check for contamination as well.</dd> |
</dl> | </dl> | ||
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<div id="toggletwo" > | <div id="toggletwo" > | ||
<p><b>Theory</b><br/> | <p><b>Theory</b><br/> | ||
− | The Gibson assembly | + | The Gibson assembly as described by Gibson et al., is a rapid DNA assembly method which assures directionional cloning of fragments in one single reaction. For the Gibson assembly to happen, three essential enzymes are needed: a mesophylic nuclease, a thermophylic ligase and a high fidelity polymerase. For this reaction, we used NEBuilder. In the first step of this reaction, the exonuclease rapidly cleaves off the 5’ DNA ends. The exonuclease is unstable at 50°C and gets degraded. In the second step, the designed sequence overlaps start to anneal.The polymerase then fills in the gaps. In the final step, the ligases covalently joins both ends. After this, the plasmid is ready to be transformed. This text was based on <a href="https://www.idtdna.com/pages/docs/default-source/user-guides-and-protocols/gibson-assembly.pdf?sfvrsn=16">the IDT website as seen on 13/09/2015</a></p> |
<div class="center"> | <div class="center"> |
Revision as of 13:25, 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