Difference between revisions of "Team:CPU CHINA/Experiments"

 
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         <h2>Characterization</h2>
 
         <h2>Characterization</h2>
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         <h4>You can click on these arrows to read the details of our experiments.</h4>
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         <h4>You can click on these arrows to read the protocol .</h4>
 
<center>
 
<center>
 
<div>
 
<div>
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</center>
 
</center>
  
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         <center><h4 style="font-size:unset !important">You can <a href="javascript:void(0);" onclick="showExperiment(5)">click here to see our Promoter</a>.</h4></center>
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         <center><h4 style="font-size:unset !important">You can <a href="javascript:void(0);" onclick="showExperiment(5)">click here to see the protocol of promoter identification</a>.</h4></center>
 
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"<br><br>"+
 
"<br><br>"+
 
"<h4>(1)The concentration of MTT (Biofoxx, Germany) is 5mg/ml. We prepare this by dissolving 0.5 g of MTT in 100 ml of phosphate buffer solution (PBS) or culture medium without phenol red. </h4>"+
 
"<h4>(1)The concentration of MTT (Biofoxx, Germany) is 5mg/ml. We prepare this by dissolving 0.5 g of MTT in 100 ml of phosphate buffer solution (PBS) or culture medium without phenol red. </h4>"+
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"<h4>(2)Filter the solution with 0.22 μm filter membrane to remove the bacteria, then store it at 4 ℃ (light should be avoided). Containers should be wrapped in aluminum foil during preparation and preservation.</h4>"
+
"<h4>(2)Filter the solution with 0.22 μm filter membrane to remove the bacteria, then store it at 4 ℃ (light should be avoided). Containers should be wrapped in aluminum foil during preparation and preservation.</h4>"+
 
"<br>"+
 
"<br>"+
 
"<h2>Culturing Cells </h2>"+
 
"<h2>Culturing Cells </h2>"+
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"<h2>qPCR</h2>"+
 
"<h2>qPCR</h2>"+
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"<h4>Follow the instruction in <i>Vazyme ChamQ Universal SYBR® qPCR Master Mix</i> (Vazyme Biotech, China).</h4><br>";
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"<h4>Follow the instruction in <a href=\"https://static.igem.org/mediawiki/2018/5/58/T--CPU_CHINA--experiment_qpcr2.pdf\"><i>Vazyme ChamQ Universal SYBR® qPCR Master Mix</i></a> (Vazyme Biotech, China).</h4><br>";
  
  
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var qidongzi = "<h2>Cell transfection and luciferase reporter gene assay</h2>"+
 
var qidongzi = "<h2>Cell transfection and luciferase reporter gene assay</h2>"+
 
"<h4>pGL3-Basic was used as negative Control, and pGL3-Control was used as positive Control.</h2>"+
 
"<h4>pGL3-Basic was used as negative Control, and pGL3-Control was used as positive Control.</h2>"+
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"<h4>HepG2 cells were transfected with 1ug of pGL3-htert, pGL3-hulc and pGL3-control. HepG2 cells were cultured in DEME medium containing 10% heat inactivated bovine serum at 37 ℃ and 5% CO2 constant temperature incubator. HepG2 cells were seeded in 96-well plate according to 2* 10<sup>5</sup>/ well, and cultured in non-antibiotic culture medium for 24 hours, then transfected with liposome (Lipo fectamin3000), and transfected with 3 holes per plasmid. The transfection medium was replaced after 4 to 6 h, and the DEME culture medium containing 10% calf serum was used to continue the culture. After 48 h, the cells were treated with Promege's <a><u>steady-glo Luciferase Assay System kit </u></a>and the activity of <i>Luc</i> was determined.</h4><br>";
+
"<h4>HepG2 cells were transfected with 1ug of pGL3-htert, pGL3-hulc and pGL3-control. HepG2 cells were cultured in DEME medium containing 10% heat inactivated bovine serum at 37 ℃ and 5% CO2 constant temperature incubator. HepG2 cells were seeded in 96-well plate according to 2* 10<sup>5</sup>/ well, and cultured in non-antibiotic culture medium for 24 hours, then transfected with liposome (Lipo fectamin3000), and transfected with 3 holes per plasmid. The transfection medium was replaced after 4 to 6 h, and the DEME culture medium containing 10% calf serum was used to continue the culture. After 48 h, the cells were treated with Promege's <a href=\"https://static.igem.org/mediawiki/2018/9/9a/T--CPU_CHINA--experiment_promoter.pdf\"><u>steady-glo Luciferase Assay System kit </u></a>and the activity of <i>Luc</i> was determined.</h4><br>";
 
 
 
var zhuanran = "<h2>Cell transfection and Screening</h2>"+
 
var zhuanran = "<h2>Cell transfection and Screening</h2>"+
 
"<h3>1.Cell transfection</h3>"+
 
"<h3>1.Cell transfection</h3>"+
−
"<h4>HepG2 cells were cultured in DEME medium containing 10% heat inactivated bovine serum at 37 ℃ and 5% CO2 constant temperature incubator. HepG2 cells were seeded in 96-well plate according to 2*10<sup>5</sup>/ well, and cultured in non-antibiotic culture medium for 24 hours, then transfected with liposome (<a><u>Lipofectamine 3000</u></a>) , and transfected with 3 holes per plasmid. The transfection medium was replaced after 4 to 6 h, and the DEME culture medium containing 10% calf serum was used to continue the culture. </h4><br>"+
+
"<h4>(1) Culture HepG2 cells with 10% heat inactivated bovine serum (Gibco, US) in DEME medium  and a 5% CO2 constant temperature incubator at 37 ℃.</h4><br>"+
−
+
                "<h4>(2) Seed HepG2 cells in a 96-well plate at a concentration of 2*10<sup>5</sup>/well, and culture them in non-antibiotic culture medium (Gibco, US) for 24 hours. </h4><br>"+
 +
"<h4>(3) Transfect the cell with Lipofectamine 3000 (Thermofisher, US).</h4><br>"+
 +
                "<h4>(4) Refresh the medium at 4h to 6h after the transfection with DEME culture medium containing 10% calf serum (Gibco, US). </h4><br>"+
 
"<h3>2.Screening</h3>"+
 
"<h3>2.Screening</h3>"+
−
"<h4>After 24-48 h, the DEME culture medium containing antibiotics (Neomycin) was used to screen.</h4><br>"+
+
"<h4>After 24-48 h, use the DEME culture medium containing antibiotics (Neomycin) (Gibco, US) for the screening of cells successfully transfected with plasmids. </h4><br>"+
 
 
 
"<h2>Luciferase reporter gene assay</h2>"+
 
"<h2>Luciferase reporter gene assay</h2>"+
−
"<h4>pGL3-Basic was used as negative Control, and pGL3-Control was used as positive Control. HepG2 cells were transfected with 1ug of pGL3-htert, pGL3-hulc and pGL3-control(Refer Cell transfection and Screening-Part.1).<h4>"+
+
"<h4>After 48 h, follow the instruction in <a href=\"https://static.igem.org/mediawiki/2018/9/9a/T--CPU_CHINA--experiment_promoter.pdf\"><i>steady-glo Luciferase Assay System kit</i></a> (Promege, US) to measure the activity of luciferase expressed from the plasmids. </h4><br>";
−
"<h4>After 48 h, the cells were treated with Promege's <a><u>steady-glo Luciferase Assay System kit </u></a>and the activity of <i>Luc</i> was determined.</h4><br>";
+
 
 
 
 

Latest revision as of 15:09, 7 December 2018

Experiments

Molecular Cloning

Transfection

You can click on the figure to see the protocol.

Characterization

You can click on these arrows to read the protocol .

You can click here to see the protocol of promoter identification.