Team:Slovenia/Notebook

From 2012.igem.org

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<li>The purity and concentration of the isolated DNA was analysed using NanoDrop. </li>
<li>The purity and concentration of the isolated DNA was analysed using NanoDrop. </li>
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<h3>Fragment DNA isolation from agarose gel</h3>
<h3>Fragment DNA isolation from agarose gel</h3>
<p><b>AGAROSE ELECTROPHORESIS</b></p>
<p><b>AGAROSE ELECTROPHORESIS</b></p>
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<h3>RESTRICTION DIGEST</h3>
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<h3>Restriction digest</h3>
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<li>To digest the desired DNA restriction reactions were prepared as follows: </li>
<li>To digest the desired DNA restriction reactions were prepared as follows: </li>
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<h3>PCR REACTION</h3>
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<h3>PCR reaction</h3>
<p>AccuPrime and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase. </p>
<p>AccuPrime and Phusion DNA polymerase were used for DNA amplification. Colony PCR was performed with Taq DNA polymerase. </p>
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<h3>Ligation</h3>
<h3>Ligation</h3>
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p>
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p>
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<h3> Culturing bacteria</h3>
<h3> Culturing bacteria</h3>
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p>
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p>
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<p><b> Luria Broth (LB) </b>: 10 g/L tryptone,    5 g/L yeast extract,    10 g/L NaCl,    media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p>
<p><b> Luria Broth (LB) </b>: 10 g/L tryptone,    5 g/L yeast extract,    10 g/L NaCl,    media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p>
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p>
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p>
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  <h3>Transformation of bacteria</h3>
  <h3>Transformation of bacteria</h3>
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  <h3>Glycerol stock for long term storage of bacteria</h3>
  <h3>Glycerol stock for long term storage of bacteria</h3>
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The induction systems are described <a href="https://2012.igem.org/Team:Slovenia/Parts">here</a>.  
The induction systems are described <a href="https://2012.igem.org/Team:Slovenia/Parts">here</a>.  
<p>The induction systems are described here. </p>
<p>The induction systems are described here. </p>
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<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li>
<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details). </li>
<li>2 hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li>
<li>2 hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v). </li>
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<li>After 2-3 days of incubation, replace cell medium with fresh medium and stimulate again appropriately. </li>
 
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<h3>Induction of cells</h3>
 
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<li>Transfect HEK293 or HEK293T cells with plasmids using JetPei transfection reagent (Polyplus transfection), following the manufacturers protocol (see cell culturing for details).</li>
 
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<li>2 hours post transfection change media and stimulate the cells by adding dilutions of appropriate inductors to the medium in a 1:10 (v:v).</li>
 
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<table class="normal" style="font-size:90%; width:90%; text-align:center;">
<table class="normal" style="font-size:90%; width:90%; text-align:center;">
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<h2>Flow cytometry </h2>
<h2>Flow cytometry </h2>
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p>
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p>

Revision as of 18:51, 26 September 2012