Difference between revisions of "Team:KU Leuven/Research/Methods"

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<h3> Methodology</h3></br>
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<div class="example">
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<div class="one">
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<h2>Preparing electrocompetent cells</h2>
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</div>
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<div id="one" div style="text-align:left; margin:20px">
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Make a liquid culture of a single colony in 1-3 mL salt free LB </br>
 +
Grow 300-400 mL cells (without salt) in 37°C until the O.D.reaches 0.6</br>
 +
Cool down on ice and from now on perform all the steps at 4 °C</br>
 +
Spin the cells down in falcon tubes (3500 g, 20 min, 4°C)</br>
 +
Resuspend the cells in 10 % glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times</br>
 +
Resuspend the cells in 10 % glycerol to obtain a dense pulp (usually not more than 1.5 mL)</br>
 +
Take 50 µL sample and do the electroporation test (without DNA). You should have a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL glycerol</br>
 +
Aliquot the cells (50 µL) and quick-freeze in liquid nitrogen and store at -80 °C</br>
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</div>
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</div>
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</br>
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<div class="example">
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<div class="two">
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<h2>Electroporation</h2>
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</div>
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<div id="two" div style="text-align:left; margin:20px;">
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Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)</br>
 +
Electroporate (Eppendorf, 1700 V, 4 msec)</br>
 +
Add 950 µl of SOC solution</br>
 +
Incubate for one hour at 37 °C</br>
 +
Plate this out on pre-warmed plates (37 °C)</br>
 +
J23101, J23106 and J23117 were plated out on chloramphenicol and I13504 was plated out on ampicillin</br>
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</div>
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</div>
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</br>
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<div class="example">
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<div class="three">
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<h2>This is example three</h2>
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<div id="three" div style="text-align:left; margin:20px">
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tiralalalala <br/>
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tiralalala <br/>
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<h2>This is example four</h2>
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tiralalalala <br/>
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tiralalala <br/>
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<div class="five">
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<h2>This is example five</h2>
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<div id="five"div style="text-align:left; margin:20px;">
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tiralalalala <br/>
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tiralalala <br/>
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<h2>Some amazing header<span> doop</span></h2>
 
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<p>Nihilne te nocturnum praesidium Palati, nihil urbis vigiliae. Cum sociis natoque penatibus et magnis dis parturient. Unam incolunt Belgae, aliam Aquitani, tertiam. Hi omnes lingua, institutis, legibus inter se differunt.</p>
 
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<p>Curabitur blandit tempus ardua ridiculus sed magna. Quam diu etiam furor iste tuus nos eludet? Curabitur est gravida et libero vitae dictum. Qui ipsorum lingua Celtae, nostra Galli appellantur. Hi omnes lingua, institutis, legibus inter se differunt. Curabitur blandit tempus ardua ridiculus sed magna.</p>
 
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<p>Hi omnes lingua, institutis, legibus inter se differunt. Vivamus sagittis lacus vel augue laoreet rutrum faucibus. Curabitur blandit tempus ardua ridiculus sed magna. Salutantibus vitae elit libero, a pharetra augue.</p>
 
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<h2>And yet another...</h2>
 
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<p>Curabitur blandit tempus ardua ridiculus sed magna. Inmensae subtilitatis, obscuris et malesuada fames. Vivamus sagittis lacus vel augue laoreet rutrum faucibus. Unam incolunt Belgae, aliam Aquitani, tertiam. Pellentesque habitant morbi tristique senectus et netus. Qui ipsorum lingua Celtae, nostra Galli appellantur.</p>
 
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<h2>Some amazing header</h2>
 
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<p id="para">Nihilne te nocturnum praesidium Palati, nihil urbis vigiliae. Cum sociis natoque penatibus et magnis dis parturient. Unam incolunt Belgae, aliam Aquitani, tertiam. Hi omnes lingua, institutis, legibus inter se differunt.</p>
 
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<p>Curabitur blandit tempus ardua ridiculus sed magna. Quam diu etiam furor iste tuus nos eludet? Curabitur est gravida et libero vitae dictum. Qui ipsorum lingua Celtae, nostra Galli appellantur. Hi omnes lingua, institutis, legibus inter se differunt. Curabitur blandit tempus ardua ridiculus sed magna.</p>
 
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<p>Hi omnes lingua, institutis, legibus inter se differunt. Vivamus sagittis lacus vel augue laoreet rutrum faucibus. Curabitur blandit tempus ardua ridiculus sed magna. Salutantibus vitae elit libero, a pharetra augue.</p>
 
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<h2>And yet another...</h2>
 
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<p>Curabitur blandit tempus ardua ridiculus sed magna. Inmensae subtilitatis, obscuris et malesuada fames. Vivamus sagittis lacus vel augue laoreet rutrum faucibus. Unam incolunt Belgae, aliam Aquitani, tertiam. Pellentesque habitant morbi tristique senectus et netus. Qui ipsorum lingua Celtae, nostra Galli appellantur.</p>
 
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Revision as of 17:16, 10 September 2015

Logo

Methods

  • P1 transduction


  • Gibson Assembly


  • Miniprep


  • Gel purification


  • Chemocompetent cells


  • Electrocompetent cells


  • Transformation

Methodology


Preparing electrocompetent cells

Make a liquid culture of a single colony in 1-3 mL salt free LB
Grow 300-400 mL cells (without salt) in 37°C until the O.D.reaches 0.6
Cool down on ice and from now on perform all the steps at 4 °C
Spin the cells down in falcon tubes (3500 g, 20 min, 4°C)
Resuspend the cells in 10 % glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times
Resuspend the cells in 10 % glycerol to obtain a dense pulp (usually not more than 1.5 mL)
Take 50 µL sample and do the electroporation test (without DNA). You should have a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL glycerol
Aliquot the cells (50 µL) and quick-freeze in liquid nitrogen and store at -80 °C

Electroporation

Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)
Electroporate (Eppendorf, 1700 V, 4 msec)
Add 950 µl of SOC solution
Incubate for one hour at 37 °C
Plate this out on pre-warmed plates (37 °C)
J23101, J23106 and J23117 were plated out on chloramphenicol and I13504 was plated out on ampicillin

This is example three

tiralalalala
tiralalala
tiralalala

This is example four

tiralalalala
tiralalala
tiralalala

This is example five

tiralalalala
tiralalala
tiralalala