Team:Slovenia/Notebook

From 2012.igem.org

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<h3>Transformation of bacteria</h3>
<h3>Transformation of bacteria</h3>
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For enrichment of vectors E. coli DH5alpha and TOP10 were used.
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<p> E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p>
<ol>
<ol>
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<li>100 µL of competent cells were thawed on ice.</li>
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<li>100 µL of competent cells were thawed on ice. </li>
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<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). </li>
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<li>50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution).</li>
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<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. </li>
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<li>The mixture was heat-shocked for 3 min at 42 °C. </li>
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<li>A mixture of cells and DNA solution was incubated on ice for 30-60 minutes.</li>
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<li>Cooled for 3 minute on ice. </li>
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<li> 500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. </li>
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<li>Than the mixture was heat-shocked for 45 seconds at 42 °C.</li>
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<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li>
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<li>Cooled down for 3 minute on ice.</li>
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<li>The mixture were than rescued by adding 500 µL preheated antibiotic free LB-medium and incubated for one hour at 37 °C while shaking for induction of the antibiotic resistance.</li>
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<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates.</li>
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</ol>
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<h3>Glycerol stock for long term storage of bacteria</h3>
<h3>Glycerol stock for long term storage of bacteria</h3>
<ol>
<ol>
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<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube.</li>
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<li>1 mL of an overnight culture was added to 150 µL of 80% glycerol into a cryo-tube. </li>
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<li>Mixed and incubated at room temperature for 30 min. </li>
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<li>Mixed and incubated at room temperature for 30 min.</li>
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<li>Afterwards the glycerol stock was stored at -80 °C. </li>
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<li>Afterwards the glycerol stock was stored at -80 °C.</li>
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</ol>
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<h2> Cell cultures </h2>
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<h2>Cell cultures</h2>
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<h3>Eucaryotic cell lines and cultivation</h3>
<h3>Eucaryotic cell lines and cultivation</h3>
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<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS.
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<p><b>HEK293</b> is a human cell line derived from kidney cells and grows in a monolayer culture. Cells were grown in DMEM medium supplemented with 10% FBS. </p>
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</p><p>
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<p><b>HEK293T</b> cell line is derived form HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p>
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<b>HEK293T</b> cell line is derived form HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS.
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<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p>
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<h3>Subculturing monolayer cell cultures</h3>
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<b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.
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<h3>Cultivation</h3>
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<b>A. SUBCULTURING MONOLAYER CELL CULTURES</b>
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<ol>
<ol>
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<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells.</li>
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<li>Remove and discard culture medium from a T-75 flask containing a monolayer of HEK293 or HEK293T cells. </li>
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<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer. </li>
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<li>Rinse the T-75 flask with 10 ml of PBS buffer to remove all traces of growth medium (DMEM + 10% FBS) which otherwise inhibits trypsin function. Remove and discard the PBS buffer.</li>
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<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 min. </li>
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<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting. </li>
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<li>Add 2-3 ml of trypsine solution and gently tilt the flask to ensure the trypsine solution covers all the cells. Incubate the cells in trypsin for 0,5 - 3 min.</li>
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<li>Transfer the cell suspension to a 15 ml centrifuge tube. </li>
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<li>Centrifuge the cell suspension for 5 min at 1200 rpm. </li>
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<li>When the cells start to detach from the surface, add 7 ml of growth medium to the trypsin solution. Resuspend all remaining cells from the bottom of the T-75 flask by pipetting.</li>
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<li>Remove the trypsin-containing medium from the centrifuge tube. </li>
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<li>Resuspend the cell pellet in fresh medium. </li>
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<li>Transfer the cell suspension to a 15 ml centrifuge tube.</li>
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<li>Take as much cells as you need and add fresh medium to a total volume of 10 ml. </li>
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<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li>
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<li>Centrifuge the cell suspension for 5 min at 1200 rpm.</li>
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</ol>
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<p><b>Cell plating</b><p>
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<li>Remove the trypsin-containing medium from the centrifuge tube.</li>
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<ol>
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<li>Count cells. </li>
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<li>Resuspend the cell pellet in fresh medium.</li>
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<li>Calculate the desired number of cells per well. Dilute cells in DMEM with 10% FBS. </li>
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<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li>
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<li>Take as much cells as you need and add fresh medium to a total volume of 10 ml.</li>
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<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2).</li>
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</ol>
</ol>
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<b>B. CELL PLATING</b>
 
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<ol>
 
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<li>Count cells.</li>
 
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<li>Calculate the desired number of cells per well. Dilute cells in DMEM with 10% FBS.</li>
 
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<li>Transfer the cells into an appropriate plate and place in a cell culture incubator.</li>
 
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Revision as of 16:39, 26 September 2012