Difference between revisions of "Team:Vilnius-Lithuania/Methods"

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                         <h4 class="panel-title">2. DNA electrophoresis</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
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                         <div class="panel-body">
 
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<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
   Agarose gel<br />
+
   4 mL of LB medium<br />
−
   DNA ladder<br />
+
   Antibiotic<br />
−
   6x Loading Dye<br />
+
   Transformed bacteria<br />
−
  1x TAE buffer<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
The agarose gel is loaded into electrophoresis sytem, filled with the buffer. The DNA ladder and DNA samples are then loaded into the wells. Run the gel at 130V for 30-40 minutes.
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
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<!-- MethodBox -->
 
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                 <div class="panel panel-default" style="position: relative">
 
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−
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                         <h4 class="panel-title">3. Transformation</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
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                         <div class="panel-body">
 
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<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
Competent cells<br />
+
  4 mL of LB medium<br />
−
Ligate/plasmid DNA<br />
+
  Antibiotic<br />
−
LB medium<br />
+
  Transformed bacteria<br />
−
Plates with LB medium and antibiotic<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>In order to perform transformation, the prepared competent cells are centrifuged (2 minutes, 6000 rpm, 4ºC) and most of the supernantant is discarded, leaving about 100 μL of solution, in which cells are suspended.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
−
 
+
−
<p style="margin: auto; height: auto; clear: both;">These cells are mixed with 10-20 ng of ligate or plasmid DNA. The mixture is incubated on ice for half an hour and right after the eppendorf tubes are placed in a thermostat in 42ºC for 1 minute. This is why it is called heat-schock transformation.</p>
+
−
 
+
−
<p>1 mL of LB liquid medium (without antibiotic) is added to the eppendorf tube with the mixture and grown in a shaking thermostat for an hour at 37ºC. After the tubes are pelleted by centrifuging, supernantant is discarded, leaving about 100 μL, in which cells are suspended.</p>
+
−
 
+
−
<p>Transformants are placed onto a Petri dish with LB medium and specific antibiotic. Plates are incubated in a thermostat at 37ºC for a night.</p>
+
 
</div>
 
</div>
 
</div>
 
</div>
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                 <div class="panel panel-default" style="position: relative">
 
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                         <h4 class="panel-title">4. Preparation of competent cells</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="4" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
LB medium<br />
+
  4 mL of LB medium<br />
−
NaCl solution<br />
+
  Antibiotic<br />
−
CaCl<sub>2</sub> solution<br />
+
  Transformed bacteria<br />
−
Bacterial cells<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>Grow 4 mL LB medium with the cells for 2-3 hours at 37ºC in the shaker. The cells are grown to OD<sub>600</sub> of 0.4-0.5. All the further steps are done on ice. Firstly, the cells are transferred into eppendorf tubes and pelleted in a centrifuge for 2 minutes at 6000 rpm at 4ºC. Supernatant is discarded and bacterial cells are suspended in 1 mL of NaCl solution. Tubes are centifuged once again for 2 minutes, 6000 rpm, 4ºC. Supernantant is discarded and cells are resuspended in 1 mL of CaCl<sub>2</sub> solution. They are left on ice for an hour or longer.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
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+
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                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#5">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">5. Protein expression</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="5" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
BL21(DE3) bacteria cells<br />
+
  4 mL of LB medium<br />
−
LB medium<br />
+
  Antibiotic<br />
−
specific antibiotic<br />  
+
  Transformed bacteria<br />
−
IPTG (1mM)<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>Transform plasmid DNA into BL21 (DE3) bacteria and grow overnight at 37ºC on a Petri dish with specific antibiotic. The next day inoculate bacteria to 4 mL of LB medium and grow overnight. On the third day, transfer the night culture into a few tubes with 20 mL LB medium at a dilution 1:50 and grow for 3 hours in 37ºC (until OD<sub>600</sub>  ~ 0.6). Add 20 μL of IPTG (1mM) into each tube. Then half of bacteria are incubated for 3 h in 37ºC, the other half is incubated for 16 h in 16ºC.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
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+
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+
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+
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#6">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">6. NDS-PAGE</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="6" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
 
+
  4 mL of LB medium<br />
 +
  Antibiotic<br />
 +
  Transformed bacteria<br />
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p></p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
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                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
<!-- /MethodBox -->
+
<!-- /MethodBox --><!-- MethodBox -->
−
 
+
−
<!-- MethodBox -->
+
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#7">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">7. Western blot</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="7" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
 
+
  4 mL of LB medium<br />
 +
  Antibiotic<br />
 +
  Transformed bacteria<br />
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p></p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
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                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
<!-- /MethodBox -->
+
<!-- /MethodBox --><!-- MethodBox -->
−
 
+
−
<!-- MethodBox -->
+
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#8">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">8. Mutagenesis</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="8" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
−
<div class="col-md-12">
+
  <div class="col-md-4">
−
   Mutagenesis was performed according to manufacturer's <a href="https://static.igem.org/mediawiki/2015/9/93/Vilnius15_mutagenesiskit.pdf">instructions.</a><br />
+
   <strong>Materials:</strong><br />
−
 
+
  4 mL of LB medium<br />
 +
  Antibiotic<br />
 +
  Transformed bacteria<br />
 
   </div>
 
   </div>
−
<p></p>
+
  <div class="col-md-8">
 +
  <strong>Process</strong>:<br />
 +
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
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                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
<!-- /MethodBox -->
+
<!-- /MethodBox --><!-- MethodBox -->
−
 
+
−
<!-- MethodBox -->
+
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#9">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">9. Gel extraction</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="9" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
−
   <div class="col-md-12">
+
   <div class="col-md-4">
−
Gel extraction was performed according to manufacturer's <a href="https://static.igem.org/mediawiki/2015/8/8b/Vilnius15_GelExtraction.pdf">instructions.</a><br />
+
  <strong>Materials:</strong><br />
−
 
+
  4 mL of LB medium<br />
 +
  Antibiotic<br />
 +
  Transformed bacteria<br />
 
   </div>
 
   </div>
−
<p></p>
+
  <div class="col-md-8">
 +
  <strong>Process</strong>:<br />
 +
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
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                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
<!-- /MethodBox -->
+
<!-- /MethodBox --><!-- MethodBox -->
−
 
+
−
<!-- MethodBox -->
+
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#10">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">10. PCR purification</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="10" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
−
   <div class="col-md-12">
+
   <div class="col-md-4">
−
PCR purification was performed according to manufacturer's <a href="https://static.igem.org/mediawiki/2015/b/b7/Vilnius15_PCRPurification.pdf">instructions.</a><br />
+
  <strong>Materials:</strong><br />
−
 
+
  4 mL of LB medium<br />
 +
  Antibiotic<br />
 +
  Transformed bacteria<br />
 
   </div>
 
   </div>
−
<p></p>
+
  <div class="col-md-8">
 +
  <strong>Process</strong>:<br />
 +
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
Line 292: Line 282:
 
                 </div>
 
                 </div>
 
<!-- /MethodBox -->
 
<!-- /MethodBox -->
−
 
 
<!-- MethodBox -->
 
<!-- MethodBox -->
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#11">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">11. Preparation of electro-competent cells</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="11" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
JM107 bacteria<br />
+
  4 mL of LB medium<br />
−
LB medium<br />
+
  Antibiotic<br />
−
Milli-Q H<sub>2</sub>O<br />
+
  Transformed bacteria<br />
−
Glycerol solution (1/2 glycerol, 1/2 H<sub>2</sub>O)<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>Inoculate LB medium with 1 mL of JM107 bacteria. Inoculate 500 mL of LB medium with 500 μL of JM107 bacteria and grow until  OD<sub>600</sub> of 0.5–0.7.  Once the bacteria has reached the desired OD, they are taken out of incubation and put on ice for 15 minutes. Starting from this moment, all the steps are done on ice or in a cold temperature (4ºC). Tube is centifuged for half an hour in 3000 G and washed with distilled autoclaved water. Afterwards, they are once again centifuged for half an hour in 3000 G. This process (washing and centrifuging) is repeated for 5 times. After the last spin, bacteria is washed with glycerol solution. 100 μL of aliquot is pipetted into the tubes and ready for transformation.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
Line 318: Line 306:
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
<!-- /MethodBox -->
+
<!-- /MethodBox --><!-- MethodBox -->
−
 
+
−
<!-- MethodBox -->
+
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#12">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">12. Transformation (using electroporation)</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="12" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
Electroporator<br />
+
  4 mL of LB medium<br />
−
Electro-competent cells<br />
+
  Antibiotic<br />
−
Plates with LB medium and specific antibiotic<br />
+
  Transformed bacteria<br />
−
Ligate/plasmid DNA<br />
+
−
 
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>Turn on electroporator and optimize it for used bacteria strain. Place 1 mm electroporation cuvettes on ice. Add 1 μL of ligate/plasmid DNA solution to the cells in each of the microcentrifuge tubes. Transfer this mixture to the cold cuvette, tap on countertop two times, wipe water from exterior of cuvette and place in the electroporation module and  press pulse. Add 1 mL of 37ºC LB medium, mix up by pipetting up and down and transfer to a 15 mL falcon tube. Shake in the incubator for one hour. Finally, place 10 μL of mixture on one LB-antibiotic plate, 100 μL on another and 200 μL on third and incubate them overnight at 37ºC.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
Line 346: Line 330:
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
<!-- /MethodBox -->
+
<!-- /MethodBox --><!-- MethodBox -->
−
 
+
−
<!-- MethodBox -->
+
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#13">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">13. PCR</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="13" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
DreamTaq polymerase<br />
+
  4 mL of LB medium<br />
−
Long PCR polymerase<br />
+
  Antibiotic<br />
−
Phusion polymerase<br />
+
  Transformed bacteria<br />
−
Primers<br />
+
−
10x PCR buffer<br />
+
−
dNTP<br />
+
−
Milli-Q H<sub>2</sub>O<br />
+
−
DNA<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>All the reaction components are mixed on ice in specific proportions (adding polymerase last) and quickly transferred into preheated thermocycler.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
Line 378: Line 355:
 
                 </div>
 
                 </div>
 
<!-- /MethodBox -->
 
<!-- /MethodBox -->
−
 
 
<!-- MethodBox -->
 
<!-- MethodBox -->
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#14">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">14. Colony PCR</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="14" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
DreamTaq polymerase<br />
+
  4 mL of LB medium<br />
−
Phusion polymerase<br />
+
  Antibiotic<br />
−
dNTP<br />
+
  Transformed bacteria<br />
−
Primers<br />
+
−
10x PCR buffer<br />
+
−
Milli-Q H<sub>2</sub>O<br />
+
−
Bacteria from colony<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>All the reaction components, except the bacteria, are mixed on ice in specific proportions (adding polymerase last). To each PCR tube, containing the PCR reaction, the small amount of bacteria from colony is added. This is made with a wooden stick and each colony is marked on the plate, as well as transferred onto a new plate. The PCR tubes are quickly transferred into a preheated thermocycler.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
Line 408: Line 380:
 
                 </div>
 
                 </div>
 
<!-- /MethodBox -->
 
<!-- /MethodBox -->
−
 
 
<!-- MethodBox -->
 
<!-- MethodBox -->
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#15">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">15. Restriction</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="15" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
Restriction enzymes<br />
+
  4 mL of LB medium<br />
−
10x Fast Digest buffer<br />
+
  Antibiotic<br />
−
FastAP<br />
+
  Transformed bacteria<br />
−
DNA<br />
+
−
Milli-Q H<sub>2</sub>O<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>All the reaction components are mixed on ice, adding the restriction enzymes last. Tubes are incubated at 37ºC for half an hour.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
Line 435: Line 404:
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
−
<!-- /MethodBox -->
+
<!-- /MethodBox --><!-- MethodBox -->
−
 
+
−
<!-- MethodBox -->
+
 
                 <div class="panel panel-default" style="position: relative">
 
                 <div class="panel panel-default" style="position: relative">
 
                     <div class="panel-heading">
 
                     <div class="panel-heading">
−
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#16">
+
                         <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#1">
−
                         <h4 class="panel-title">16. Ligation</h4>
+
                         <h4 class="panel-title">1. Plasmid DNA extraction</h4>
 
                         </a>
 
                         </a>
 
                     </div>
 
                     </div>
−
                     <div id="16" class="panel-collapse collapse">
+
                     <div id="1" class="panel-collapse collapse">
 
                         <div class="panel-body">
 
                         <div class="panel-body">
 
<div class="row">
 
<div class="row">
 
   <div class="col-md-4">
 
   <div class="col-md-4">
 
   <strong>Materials:</strong><br />
 
   <strong>Materials:</strong><br />
−
T4 Ligase<br />
+
  4 mL of LB medium<br />
−
10x T4 Ligase buffer<br />
+
  Antibiotic<br />
−
Vector DNA<br />
+
  Transformed bacteria<br />
−
Plasmid DNA<br />
+
−
Milli-Q H<sub>2</sub>O<br />
+
 
   </div>
 
   </div>
 
   <div class="col-md-8">
 
   <div class="col-md-8">
 
   <strong>Process</strong>:<br />
 
   <strong>Process</strong>:<br />
−
<p>All the reaction components are mixed on ice, adding the ligase enzyme last. Tubes are incubated at room temperature for 10-15 minutes. Then ligase enzyme is inactivated by incubating tubes at 65ºC for 10 minutes. Afterwards, ligation reaction is chilled on ice and used in transformation.</p>
+
  Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's <a href="https://static.igem.org/mediawiki/2015/b/b1/Vilnius15_PlasmidMiniprep.pdf">instructions.</a>
 
</div>
 
</div>
 
</div>
 
</div>
Line 464: Line 429:
 
                 </div>
 
                 </div>
 
<!-- /MethodBox -->
 
<!-- /MethodBox -->
−
 
−
 
−
 
−
 
  
 
</section>
 
</section>

Revision as of 17:08, 7 September 2015

Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.
Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria
Process:
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions.