Team:Braunschweig/Protocols

From 2013.igem.org

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<div id="Gelelectrophoresis" class="menuSection">
<div id="Gelelectrophoresis" class="menuSection">
     <h2><a href="#Gelelectrophoresis">Gel Electrophoresis</a></h2>
     <h2><a href="#Gelelectrophoresis">Gel Electrophoresis</a></h2>
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     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:0px;">Coming soon</p>
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     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Depending on the size of the DNA fragments a 0.8 to 2% agarose gel with 0.2 µg/mL ethidiumbromide was used. The gel was placed in an electrophoresis chamber and covered in 1x TAE buffer. Subsequently DNA samples mixed with loading buffer were loaded to the gel pockets and separated at 80-120 V for about 2 h. Last the gel was photographed for documentation.</p>
    
    
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<div id="Cloning" class="menuSection">
<div id="Cloning" class="menuSection">
     <h2><a href="#Cloning">Cloning</a></h2>
     <h2><a href="#Cloning">Cloning</a></h2>
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     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:0px;">Coming soon</p>
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     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:0px;">
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<b>Restriction digest</b><br>
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For restriction of vector and insert DNA ca. 1000 ng purified DNA, 5 µL 10x buffer and 1 µL of each of the two restriction enzymes were mixed in a 1.5 mL reaction tube. Water was added to a final volume of 50 µL. Restriction was carried out at 37 °C for 1 h. The reaction was stopped by inactivating the enzymes at 80 °C for 20 min.<br>
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The buffer was chosen depending on the combination of restriction enzymes used:<br>
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- PstI/EcoRI HF: NE 2.1<br>
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- PstI/SpeI HF: NE 2.1<br> 
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- PstI/XbaI: NE 3.1<br>
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- all other combinations: NEB CutSmart Buffer<br><br>
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<b>Dephosphorylation</b><br>
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For the desphosphorylation of vector DNA 5.56 µL Antarctic Phos Reaction Buffer (NEB) and 0.5µL Antarctic Phosphatase (NEB) were added to the digestet DNA and mixed. DNA was incubated at 37 °C for 60 min. After 30 min of incubation 0.5 µL Antarctic Phosphatase was added to the DNA and mixed.<br><br>
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<b>DNA Purification</b><br>
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After desphosphorylation the vector DNA was purified using the Macherey-Nagel Nucleospin Gel and PCR Clean-up Kit.<br><br>
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<b>Ligation</b><br>
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For ligation of the constructed plasmid 50 ng vector DNA, 3 times as much insert DNA, 2 µL T4 Ligase Buffer (NEB), 0.5 µL T4 Ligase (NEB) and water added to a final volume of 20 µL were mixed in 1.5 mL reaction tube. Ligation was carried out at 16 °C over night. The reaction was stopped by inactivating the enzymes at 65 °C for 20 min.<br><br>
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<b>Transformation of chemocompetent cells via heatshock</b><br>
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One -80°C glycerol-stock of chemocompetend cells was thawed on ice. 10 µL of the ligated DNA was prepared in a 1.5 mL reaction tube. 50 µL of the cells were added and incubated for 20 min on ice. The mixture was then heat shocked at 42 °C for 1 min and cooled on ice for 2 min. 1 mL S.O.C.-medium was added and the cells were icubated for 1 h at 37 °C while shaken at 600 rpm.
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100 µL of the cell suspension were plated on a 2xYT agar plate with the according antibiotic (Chloramphenicol or Ampicillin) and incubated at 37 °C over night.<br><br>
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Revision as of 00:08, 26 September 2013

Protocols

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In this section you will find detailed protocols of experimental procedures.

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