Difference between revisions of "Team:Goettingen/Notebook"

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<h2>Notebook</h2>
 
<h2>Notebook</h2>
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<p> Document the dates you worked on your project.</p>
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<p><h1><strong>LB medium</strong></h1></p>
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<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Add the following components</p>
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<table border="1" cellspacing="0" cellpadding="0">
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<tbody>
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<tr>
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<td valign="top" width="109">
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<p>NaCl</p>
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</td>
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<td valign="top" width="150">
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<p>10 g</p>
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</td>
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</tr>
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<tr>
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<td valign="top" width="109">
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<p>Yeast extract</p>
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</td>
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<td valign="top" width="150">
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<p>5 g</p>
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</td>
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</tr>
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<tr>
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<td valign="top" width="109">
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<p>Tryptone</p>
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</td>
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<td valign="top" width="150">
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<p>10 g</p>
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</td>
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</tr>
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<tr>
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<td valign="top" width="109">
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<p>H<sub>2</sub>O</p>
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</td>
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<td valign="top" width="150">
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<p>Add to 1 L</p>
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</td>
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</tr>
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</tbody>
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</table>
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<ul>
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<li>To obtain solid media add 15g/L agar.</li>
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</ul>
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<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Autoclave at 121 <sup>o</sup>C for 20 min.</p>
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<p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; The preferred antibiotic is added with the proper concentration (e.g Ampicilin is added to a final concentration of 100 &micro;g/ml)</p>
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<ul>
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<li>To the liquid medium antibiotic is added upon usage.</li>
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<li>For the preparation of agar plates antibiotic is added after autoclaving the media and cooling it to 60 <sup>o</sup>C.&nbsp;</li>
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</ul>
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<p>&nbsp;</p>
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<p>
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    <h1><strong>Plasmid Extraction - using peqGOLD Plasmid Miniprep Kit I,
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PEQLAB Technologies</strong></h1>
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</p>
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<p>
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    Select few TOP10/BL21 <em>E.coli</em> transformed colonies from the LB Amp plates containing the propagated transformed colonies and inoculate them into
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    liquid LB medium (5 ml) containing preferred antibiotic of desired concentration in each tube.
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</p>
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<p>
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    Incubate the LB tubes with the transformed colonies at 37ᵒC in a shaker for about 12 – 15 hours (overnight) with agitation (150 rpm).
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</p>
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<p>
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    Extract the plasmids from the incubated LB cultures using the peqGOLD Plasmid Miniprep Kit I.
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</p>
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<p>
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    Centrifuge the culture at 10000 x g for 2 min to obtain the pellet and repeat the process until the culture is completely centrifuged. Store the pellet of
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    1 ml of the culture at -20ᵒC for future use.
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</p>
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<p>
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    Resuspend the pellet in 250 µl of Solution I of the Kit (which is normally kept at 4ᵒC because of the RNase) and vortex until the pellet is resuspended.
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</p>
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<p>
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    Add 250 µl of Solution II to the resuspended mixture and gently mix by inverting and rotating the tubes 6 -10 times to obtain a cleared lysate. Incubate
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    the mixture for 2 min to obtain optimum results.
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</p>
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<p>
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    Add 350 µl of Solution III to the cleared lysate and gently mix by inverting the tubes 6 -10 times until a flocculent white precipitate is formed.
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    Centrifuge at 10000 x g for 10 min at room temperature.
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</p>
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<p>
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    Transfer the clear supernatant to a fresh PerfectBind DNA Column in a 2 ml Collection Tube. Centrifuge the Column with the Collection Tube for 1 min at
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    10000 x g at room temperature. Discard the flow-through liquid.
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</p>
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<p>
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    Add 500 µl of PW Plasmid buffer to the PerfectBind DNA Column in the Collection Tube and centrifuge for 1 min at 10000 x g. Discard the flow-through.
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</p>
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<p>
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    Add 750 µl of DNA Wash buffer to the PerfectBind DNA Column in the Collection tube and centrifuge for 1 min at 10000 x g. Discard the flow-through. Repeat
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    this step to obtain optimum results.
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</p>
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<p>
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    Place the PerfectBind DNA Column in the Collection tube and centrifuge for 2 min at 10000 x g to dry the column matrix. This step is essential to remove
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    ethanol from the column.
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</p>
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<p>
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    Place the PerfectBind DNA Column into a fresh 1.5 ml Eppendorf tube. Add 50 µl of pre-warmed sterile deionized water directly to the binding matrix in the
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    PerfectBind DNA Column and centrifuge for 1 min at 5000 x g to elute the DNA.
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</p>
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<p>
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    Discard the PerfectBind DNA Column and store the eluted plasmid DNA at -20ᵒC.
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</p>
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<p>
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    Check the concentration of the plasmids by using a NanoDrop and note down the values for future experiments.
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</p>
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<p>&nbsp;</p>
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<p>
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    <h1><strong>Plasmid Extraction - using QIAprep Spin Miniprep Kit (QIAGEN)</strong></h1>
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</p>
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<p>This protocol describes the purification of plasmid DNA from 5 ml overnight cultures of <em>E. coli</em> grown in LB medium using the QIAprep Spin Miniprep Kit. (QIAGEN).</p>
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<ul>
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<li>Add the provided RNase A solution to buffer P1, mix, and store at 4 <sup>o</sup>C.</li>
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<li>Add ethanol (96&ndash;100%) to Buffer PE before use.</li>
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<li>All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.</li>
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</ul>
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<p>&nbsp;</p>
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<p>- Add the proper antibiotic with the proper concentration to 5 ml LB medium (e.g Ampicilin is added to a final concentration of 100 &micro;g/ml).</p>
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<p>&nbsp;</p>
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<p>- Inoculate the medium with the desired <em>E.coli</em> strain and incubate overnight at 37 <sup>o</sup>C with agitation (150 rpm).</p>
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<p>&nbsp;</p>
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<p>- Pellet the overnight culture by centrifugation at 8000 rpm (6800xg) for 3 min at room temperature.</p>
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<p>&nbsp;</p>
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<p>- Resuspend pelleted bacterial cells in 250 &mu;l Buffer P1 and transfer it to a microcentrifuge tube.</p>
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<p>&nbsp;</p>
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<p>- Add 250 &mu;l Buffer P2 and mix thoroughly by inverting the tube 4&ndash;6 times. Do not allow the lysis reaction to proceed for more than 5 min.</p>
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<p>&nbsp;</p>
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<p>- Add 350 &mu;l Buffer N3 and mix immediately and thoroughly by inverting the tube 4&ndash;6 times.</p>
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<p>&nbsp;</p>
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<p>- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.</p>
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<p>&nbsp;</p>
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<p>- Apply the supernatant to the QIAprep spin column by decanting. Centrifuge 60 s. Discard the flow-through.</p>
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<p>&nbsp;</p>
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<p>- Wash the QIAprep spin column by adding 500 &mu;l Buffer PB and centrifuging for 60 s. Discard the flow-through.</p>
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<p>&nbsp;</p>
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<p>- Wash QIAprep spin column by adding 750 &mu;l Buffer PE and centrifuging for 30&ndash;60 s.</p>
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<p>&nbsp;</p>
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<p>- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.</p>
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<p>&nbsp;</p>
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<p>- Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.</p>
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<p>&nbsp;</p>
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<p>- To elute DNA, add 50 &mu;l water (40 &ndash; 60 <sup>o</sup>C) to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.</p>
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<h5>What should this page have?</h5>
 
<h5>What should this page have?</h5>
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<li>Mention who participated in what task.</li>
 
<li>Mention who participated in what task.</li>
 
</ul>
 
</ul>
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<p> Document the dates you worked on your project.</p>
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<h4>Inspiration</h4>
 
<h4>Inspiration</h4>

Revision as of 19:42, 7 September 2015



Notebook

Document the dates you worked on your project.

What should this page have?
  • Chronological notes of what your team is doing.
  • Brief descriptions of daily important events.
  • Pictures of your progress.
  • Mention who participated in what task.

Inspiration

You can see what others teams have done to organize their notes: