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| | <h2> | | <h2> |
| − | Introduction
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| − | <br></br>
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| − | </p>
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| | </div> | | </div> |
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| − | <h2>
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| − | Methodology</h2>
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| − | </br>
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| − | <div class="center">
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| − | <div class="togglebar">
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| − | <div class="toggleone">
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| − | <h2>Preparing electrocompetent cells</h2>
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| − | </div>
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| − | <div id="toggleone">
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| − | <p>Make a liquid culture of a single colony in 1-3 mL salt free LB
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| − | <br/>
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| − | Grow 300-400 mL cells (without salt) at 37 °C until the O.D. reaches 0.6</br>
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| − | Cool down on ice and perform all the steps at 4 °C</br>
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| − | Spin the cells down in falcon tubes (3500 g, 20 min, 4 °C)</br>
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| − | Resuspend the cells in 10% glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times</br>
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| − | Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not more
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| − | than 1.5 mL)</br>
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| − | Take 50 µL sample and do the electroporation test (without DNA). Pulses should be
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| − | between 4 and 6 msec. If shorter, wash the cells once again with 30 mL
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| − | glycerol</br>
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| − | Aliquot the cells (50 µL), quick-freeze in liquid nitrogen and store at -80 °C</br></p>
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| − | </div>
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| − | </div>
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| − | </br>
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| − | <div class="togglebar">
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| − | <div class="toggletwo">
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| − | <h2>Electroporation</h2>
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| − | </div>
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| − | <div id="toggletwo" >
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| − | <p>Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)</br>
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| − | Electroporate (Eppendorf, 1700 V, 4 msec)</br>
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| − | Add 950 µl of SOC solution</br>
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| − | Incubate for one hour at 37 °C</br>
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| − | Plate out on pre-warmed plates containing the correct selective medium, in this case chlormaphenicol for J23101, J23106 and J23117 and ampicillin for I13504 (37 °C)</br></p>
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| − | </div>
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| − | </div>
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| − | </br>
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| − | <div class="togglebar">
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| − | <div class="togglethree">
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| − | <h2>BioBrick Assembly Method</h2>
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| − | </div>
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| − | <div id="togglethree" >
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| − | <p>Digest I13504 (GFP) with XbaI and PstI in Tango buffer</br>
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| − | Digest the promoters J23101, J23106 and J23117 with PstI in buffer O</br>
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| − | Load the digested I13504 on a 1.5% agarose gel and visualize it under UV light.
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| − | Thereafter, perform a gel purification of I13504 (GeneJET Gel Extraction Kit -
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| − | ThermoFisher Scientific)</br>
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| − | PCR purify the promoters J23101, J23106 and J23117</br>
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| − | Digest the promoters J23101, J23106 and J23117 with FD SpeI in 10x Fast Digest
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| − | Buffer</br>
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| − | Ligate every promoter with I13504 using T4 DNA ligase</br></p>
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| − | </div>
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| − | </div>
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| − | </br>
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| − | <div class="togglebar">
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| − | <div class="togglefour">
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| − | <h2>Restriction mapping</h2>
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| − | </div>
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| − | <div id="togglefour">
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| − | <p>Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in Tango buffer</br>
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| − | Mix gently and spin down</br>
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| − | Incubate for 2 hours at 37 °C in a heating block</br>
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| − | Separate the fragments using gel electrophoresis on a 1.5% agarose gel</br></p>
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| − | </div>
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| − | </div>
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| − | </br>
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| − | </div>
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| − | <div class="part">
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| − | <h2>
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| − | Worksheet
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| − | </h2>
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| − | <p>
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| − | Our wetlab team worked well together to fulfill this challenge. Vincent Van Deuren and Laurens Vandenbroek performed the BioBrick assembly and the transformation experiments. The measurements were recorded by Laetitia Van Wonterghem, Ovia Margaret Thirukkumaran and Laurens Vandenbroek. Laura Van Hese, Astrid Deryckere, Ines Cottignie and Vincent Van Deuren carried out the restriction digestion to check for the inserts. Finally, the results were processed by Ovia Margaret Thirukkumaran and Laurens Vandenbroek and our wiki-page was filled with provided data by Vincent Van Deuren.</br>
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| − |
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| − | <br> To grow our cells, we made use of a New Brunswick Innova® 43/43R Shaker purchased from Eppendorf. This incubator has a throw of 2.54 cm. Our devices were measured by a Tecan Safire2 monochromator MTP Reader. This machine was last calibrated on the 31<sup>th</sup> of March in 2015 by Tecan and our measurements took place on the 25<sup>th</sup> of August in 2015. The cells were excited at 483 nm and the emission was recorded at 525 nm. To capture the light emission, a Quad4 Monochromator was used. The absorbance was measured at 600 nm with a sampling frequency of 0.11 seconds/ sample while the sampling frequency of the fluorescence was 0.15 seconds/sample.
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| − |
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| − | </br>
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| − | </p>
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| − | </div>
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| − | </div>
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| − | </div>
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| | <!--Foot don't touch!--> | | <!--Foot don't touch!--> |
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