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Team:Paris Bettencourt/Notebook/Interlab Study
From 2014.igem.org
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<h4>July </h4> | <h4>July </h4> | ||
<h5 id="date">July 4th</h5> | <h5 id="date">July 4th</h5> | ||
| - | <p id="text"> | + | <p id="text"><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;"><div><div style="font-size: 15px;"><u>We will participate in the iGEM interlab study: https://2014.igem.org/Tracks/Measurement/Interlab_study</u></div><div><br clear="none"/></div><div>The three specific devices teams are required to measure fluorescence data for are given below.</div><div><br clear="none"/></div><div>Existing device: <strong>BBa_I20260 (J23101-B0032-E0040-B0015)</strong> in the <strong>pSB3K3</strong> vector.</div><ol><li>Kit location |
| + | <ol><li>Plate 4, Well 18A</li></ol></li></ol><div>E.Coli <strong>NEBTurbo</strong> competent cells</div><p>To use the DNA in the Distribution Kit you may follow these instructions:</p><ol><li>With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want.</li><li>Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.</li><li><a href="http://parts.igem.org/Help:Transformation_Protocol" shape="rect" target="_blank" title="Help:Transformation Protocol"><span style="color:rgb(0,102,204);"><span>Transform</span></span></a> 1 or 2uL of the resuspended DNA into your desired competent cells,</li></ol><div><u style="font-size: 15px;">Heat Shock Transformation of <em>E. coli</em></u></div><p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p><h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5><ol><li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li><li>Place 20 ul of cells in a pre-chilled Eppendorf tube. | ||
| + | <ul><li><span style="text-decoration: underline;">For an Intact Vector:</span> Add 0.5 ul or less to the chilled cells</li></ul></li><li>Mix gently by flicking the tube.</li><li>Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li><li>Heat shock at 42 °C for 30 seconds.</li><li>Return to ice for 2 minutes.</li><li>Add 200 ul LB medium and recover the cells by shaking at 37 °C.Kanamycin: 120 minutes</li><li>Plate out the cells on selective LB.Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.</li><li>Incubate at 37 °C. Transformants should appear within 12 hrs.</li></ol><div><span style="background-color:rgb(255, 250, 165);-evernote-highlight:true;"><u>Result:</u></span> Bacteria grow and made big ans small colonies</div><div><br clear="none"/></div></div><div> 4.Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.</div><div><br clear="none"/></div><div><br clear="none"/></div><div><br clear="none"/></div><div><br clear="none"/></div><div><br clear="none"/></div><div><br clear="none"/></div><br/></div></p> | ||
<p id="text">Text</p> | <p id="text">Text</p> | ||
<h5 id="date">Date 2</h5> | <h5 id="date">Date 2</h5> | ||
Revision as of 17:09, 18 August 2014
Paris Bettencourt 2014
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Notebook
Interlab Study
July
July 4th
We will participate in the iGEM interlab study: https://2014.igem.org/Tracks/Measurement/Interlab_study
The three specific devices teams are required to measure fluorescence data for are given below.
Existing device: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector.
- Kit location
- Plate 4, Well 18A
E.Coli NEBTurbo competent cells
To use the DNA in the Distribution Kit you may follow these instructions:
- With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want.
- Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.
- Transform 1 or 2uL of the resuspended DNA into your desired competent cells,
Heat Shock Transformation of E. coli
This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.
Note: Never vortex competent cells. Mix cells by gentle shaking.
- Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
- Place 20 ul of cells in a pre-chilled Eppendorf tube.
- For an Intact Vector: Add 0.5 ul or less to the chilled cells
- Mix gently by flicking the tube.
- Chill on ice for 10 minutes. This step is optional, but can improve yields when transforming a ligation product.
- Heat shock at 42 °C for 30 seconds.
- Return to ice for 2 minutes.
- Add 200 ul LB medium and recover the cells by shaking at 37 °C.Kanamycin: 120 minutes
- Plate out the cells on selective LB.Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.
- Incubate at 37 °C. Transformants should appear within 12 hrs.
Result: Bacteria grow and made big ans small colonies
4.Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
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