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

 
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<!--*****************************正文主体********************************* -->
 
<!--*****************************正文主体********************************* -->
 
<div class="container" style="position:relative;top:300px;">
 
<div class="container" style="position:relative;top:300px;">
 +
        <center><h1>Experiments</h1></center>
 +
        <h2>Molecular Cloning</h2>
 
<center>
 
<center>
 
<img style="width:80% !important;"src="https://static.igem.org/mediawiki/2018/7/7e/T--CPU_CHINA--Experiment-1.png">
 
<img style="width:80% !important;"src="https://static.igem.org/mediawiki/2018/7/7e/T--CPU_CHINA--Experiment-1.png">
 
</center>
 
</center>
−
 
+
        <h2>Transfection</h2>
 +
        <h4>You can click on the figure to see the protocol.</h4>
 
<center><a href="javascript:void(0)" onclick="showExperiment(6)"><img src="https://static.igem.org/mediawiki/2018/b/bb/T--CPU_CHINA--Experiment-2.png"></a></center>
 
<center><a href="javascript:void(0)" onclick="showExperiment(6)"><img src="https://static.igem.org/mediawiki/2018/b/bb/T--CPU_CHINA--Experiment-2.png"></a></center>
 +
 +
 +
        <h2>Characterization</h2>
 +
        <h4>You can click on these arrows to read the protocol .</h4>
 
<center>
 
<center>
 
<div>
 
<div>
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<td><img src="https://static.igem.org/mediawiki/2018/4/4f/T--CPU_CHINA--Experiment-3.png"></td>
 
<td><img src="https://static.igem.org/mediawiki/2018/4/4f/T--CPU_CHINA--Experiment-3.png"></td>
 
</tr>
 
</tr>
−
<table>
+
</table>
 
</div>
 
</div>
 
</center>
 
</center>
  
−
        <center><h4 style="font-size:unset !important">You can click on these arrows to read the details of our experiments.</h4><br></center>
+
         <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>
−
         <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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<!-- ********************************************弹窗****************************************** -->
 
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<!-- 弹窗层 -->
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</div>
 
</div>
  
−
<img class="footer" src="https://static.igem.org/mediawiki/2018/c/c9/T--CPU_CHINA--footer2.png" style="width:100%"/>
+
 
 
</body>
 
</body>
  
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var MTT="<h2>Reagent Preparation</h2>"+
 
var MTT="<h2>Reagent Preparation</h2>"+
 
"<br><br>"+
 
"<br><br>"+
−
"<h4>The concentration of MTT is 5mg/ml. Weigh MTT 0.5 g, dissolve in 100 ml of phosphate buffer solution (PBS) or culture medium without phenol red, with 0.22 μm filter membrane filtration to remove the bacteria in the solution, store at 4 ℃ and avoid light preservation. Containers are best wrapped in aluminum foil during preparation and preservation.</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>"+
 +
"<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>"+
−
"<h4>HepG2 cell (3* 10<sup>4</sup> /mL)s were seeded in 96-well plate according to 100 µL/ well, cultured at 37 ℃ and 5% CO2 constant temperature incubator.</h4>"+
+
"<h4>Seed HepG2 cells (3* 10<sup>4 </sup>/mL) with DEME medium (Gibco, US) in 96-well plate at the amount of 100 µL/ well, culture in a constant temperature incubator (37 ℃, 5% CO<sub>2</sub>).</h4>"+
 
"<br>"+
 
"<br>"+
 
"<h2>Labeling Cells</h2>"+
 
"<h2>Labeling Cells</h2>"+
−
"<h4>1. For adherent cells, remove the medium and replace it with 100 µL of fresh culture medium. For non-adherent cells, centrifuge the microplate, pellet the cells, carefully remove as much medium as possible and replace it with 100 µL of fresh medium.</h4>"+
+
"<h4>1. For adherent cells, remove the medium and replace it with 100 µL of fresh culture medium. </h4>"+
−
"<h4>2. Add 20 µL of the 12 mM MTT stock solution (prepared) to each well. Include a negative control of 20 µL of the MTT stock solution added to 100 µL of medium alone.</h4>"+
+
"<h4>2. Add 20 µL of 12 mM MTT stock solution (prepared in advance) into each well. Include a negative control of 20 µL MTT stock solution added into blank 100 µL medium.</h4>"+
−
"<h4>3. Incubate at 37°C for 4 hours. At high cell densities (>100,000 cells per well) the incubation time can be shortened to 2 hours.</h4>"+
+
"<h4>3. Incubate at 37°C for 4 hours. When cell density is high (>100,000 cells per well) the incubation time can be shortened to 2 hours.</h4>"+
−
"<h4>4. Add 150 µL of the DMSO solution to each well and mix thoroughly using the pipette.</h4>"+
+
"<h4>4. Add 150 µL of DMSO solution into each well and mix thoroughly using the pipette.</h4>"+
−
"<h4>5. Incubate the microplate at 37°C for 10 min in a humidified chamber. Longer incubations will decrease the sensitivity of the assay.</h4>"+
+
"<h4>5. Incubate the microplate at 37°C for 10 min in a humidified chamber. Lengthened incubation decrease the sensitivity of the assay.</h4>"+
−
"<h4>6. Mix each sample again using a pipette and read absorbance at 570 nm as detection wavelength and 630 nm as reference wavelength.</h4>"+
+
"<h4>6. Mix each sample again using a pipette and read its absorbance with 570 nm as the detection wavelength and 630 nm as the reference wavelength.</h4>"+
 
"<br>";
 
"<br>";
  
−
var qPCR = "<h2>The extraction of RNA</h2>"+
+
var qPCR = "<h3><i>1. Homogenization:</i></h3>"+
−
"<h3><i>A. Required reagents:</i></h3>"+
+
−
"<h4>DEPC-treated water (Ambion)、Transzol UP Reagent (Transgen)、Ice cold PBS、Cell scraper、70% ethanol、Isopropyl alcohol.</h4>"+
+
−
"<h3><i>B. Equipment and supplies:</i></h3>"+
+
−
"<h4>Refrigerated centrifuge、Microcentrifuge、Micropipettors、Aerosol-barrier tips、Vortex mixer Powder-free gloves、Centrifuge tubes.</h4>"+
+
−
"<h3><i>C. Safety:</i></h3>"+
+
−
"<h4>Always use gloves and eye protection. Avoid contact with skin or clothing. Use in a chemical hood. Avoid breathing vapor. </h4>"+
+
−
"<h3><i>D: Homogenization:</i></h3>"+
+
 
"<h4>Hepg2 cell grown in Monolayer </h4>"+
 
"<h4>Hepg2 cell grown in Monolayer </h4>"+
−
"<h4>Rinse cell monolayer with ice cold PBS once. Lyse cells directly in a culture dish by adding 1 ml of Transzol UP Reagent per 3.5 cm diameter dish and scraping with cell scraper. Pass the cell lysate several times through a pipette. Vortex thoroughly. The amount of Transzol UP Reagent added is based on the area of the culture dish (1 ml per 10 cm<sup>2</sup>) and not on the number of cells present. An insufficient amount of Transzol UP Reagent may result in DNA contamination of the isolated RNA.</h4>"+
+
"<h4>(1) Rinse the cells with ice cold PBS once. Lyse cells directly in a culture dish by adding 1 ml of Transzol UP Reagent (Transgen, China) per 3.5 cm diameter dish and scrape it with a cell scraper.</h4>"+
−
"<h3><i>E. PHASE SEPERATION:</i></h3>"+
+
"<h4>(2) Pass the cell lysate several times through a pipette. Vortex thoroughly. The amount of Transzol UP Reagent added is based on the area of the culture dish (1 ml/10 cm<sup>2</sup>) and, not on the number of cells present. An insufficient amount of Transzol UP Reagent may result in DNA contamination of the isolated RNA.</h4>"+
−
"<h4>Add 0.2 ml of chloroform per 1 ml of Transzol UP Reagent. Cap sample tubes securely. Vortex samples vigorously for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x.g for 15 minutes at 2 to 8℃. Following centrifugation, the mixture separates into lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube. Measure the volume of the aqueous phase (The volume of the aqueous phase is about 60% of the volume of Transzol UP Reagent used for homogenization).</h4>"+
+
"<h3><i>2. PHASE SEPERATION:</i></h3>"+
−
"<h3><i>F. RNA PRECIPITATION:</i></h3>"+
+
"<h4>(1) Add 0.2 ml of chloroform per 1 ml of Transzol UP Reagent. Cap sample tubes securely. Vortex samples vigorously for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 RPM for 15 minutes at 2 to 8℃. </h4>"+
−
"<h4>Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of Transzol UP Reagent used for the initial homogenization. Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000 x g for 10 minutes at 2 to 4℃. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.</h4>"+
+
"<h4>(2) Following centrifugation, a lower red phenolchloroform phase, an interphase and a colorless upper aqueous phase are visually separated within the mixture. RNA remains exclusively in the aqueous phase. </h4>"+
−
"<h3><i>G: RNA WASH:</i></h3>"+
+
"<h4>(3) Transfer the upper aqueous phase carefully without disturbing the interphase into fresh tube. Measure the volume of the aqueous phase (The volume of the aqueous phase is about 60% of the volume of Transzol UP Reagent used for homogenization).</h4>"+
−
"<h4>Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of Transzol UP Reagent used for the initial homogenization. Mix the samples by vortexing and centrifuge at no more than 7,500 x.g for 5 minutes at 2 to 8℃. Repeat above washing procedure once. Remove all leftover ethanol.</h4>"+
+
"<h3><i>3. RNA PRECIPITATION:</i></h3>"+
−
"<h3><i>H. REDISSOLVING RNA:</i></h3>"+
+
"<h4>(1) Precipitate the RNA from the aqueous phase by mixing it with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of Transzol UP Reagent used for the initial homogenization. </h4>"+
−
"<h4>Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by centrifuge under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A<sub>260</sub>/A<sub>280</sub> ratio < 1.6. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip.</h4>"+
+
"<h4>(2) Incubate samples at 15 to 30℃ for 10 minutes and centrifuge at no more than 12,000 RPM for 10 minutes at 2 to 4℃. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.</h4>"+
−
"<h3><i>I. SPECTROPHOTOMETRIC ANALYSIS:</i></h3>"+
+
"<h3><i>4: RNA WASH:</i></h3>"+
−
"<h4>Dilute 1 μl of RNA with 39 μl of DEPC-treated water (1:40 dilution). Using 10 μl microcuvette, take OD at 260 nm and 280 nm to determine sample concentration and purity. The A<sub>260</sub>/A<sub>280</sub> ratio should be above 1.6. Apply the convention that 1 OD at 260 equals 40 µg /ml RNA.</h4>"+
+
"<h4>(1) Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of Transzol UP Reagent used for the initial homogenization. </h4>"+
 +
"<h4>(2) Mix the samples by vortex and centrifugation at no more than 7,500 RPM for 5 minutes at 2 to 8℃. Repeat above washing procedure once. Remove all leftover ethanol.</h4>"+
 +
"<h3><i>5. REDISSOLVING RNA:</i></h3>"+
 +
"<h4>(1) Air-dry or vacuum dry the RNA pellet for 5-10 minutes. (Do not dry the RNA pellet by centrifuge under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility.) Partially dissolved RNA samples have an A260/A280 ratio < 1.6. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip.</h4>"+
 +
"<h3><i>6. SPECTROPHOTOMETRIC ANALYSIS:</i></h3>"+
 +
"<h4>(1) Dilute 1 μl of RNA with 39 μl of DEPC-treated water (1:40 dilution). </h4>"+
 +
"<h4>(2) Using 10 μl microcuvette, take OD at 260 nm and 280 nm to determine the sample concentration and purity. </h4>"+
 +
"<h4>(3) The A260/A280 ratio should be above 1.6. Apply the convention that 1 OD at 260 equals 40 µg /ml RNA. </h4>"+
 
"<h3>Reverse transcription</h3>"+
 
"<h3>Reverse transcription</h3>"+
−
"<h4>Use Vazyme miRNA 1st Strand cDNA Synthesis Kit (by stem-loop)</h4>"+
+
"<h4>Use Vazyme miRNA 1st Strand cDNA Synthesis Kit (by stem-loop)(Vazyme Biotech, China)</h4>"+
 
"<h4>1. Genomic DNA removal</h4>"+
 
"<h4>1. Genomic DNA removal</h4>"+
 
"<h4>a. The following mixture was prepared in the RNase-free centrifuge tube:</h4>"+
 
"<h4>a. The following mixture was prepared in the RNase-free centrifuge tube:</h4>"+
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"<td style=\"padding-left:200px !important;\"><h4>10 pg-1 μg</h4></td></tr></table>"+
 
"<td style=\"padding-left:200px !important;\"><h4>10 pg-1 μg</h4></td></tr></table>"+
  
−
"<h4>Gently blow with a pipette to mix.</h4>"+
+
"<h4>Gently blow the mixture with a pipette to mix.</h4>"+
 
"<h4>b. Genomic DNA removal was performed under the following conditions: 42℃ 2 min</h4>"+
 
"<h4>b. Genomic DNA removal was performed under the following conditions: 42℃ 2 min</h4>"+
 
"<h4>2. First strand cDNA synthesis</h4>"+
 
"<h4>2. First strand cDNA synthesis</h4>"+
Line 326: Line 333:
 
"<td style=\"padding-left:200px !important;\"><h4>2 μl</h4></td></tr></table>"+
 
"<td style=\"padding-left:200px !important;\"><h4>2 μl</h4></td></tr></table>"+
  
−
"<h4>Gently blow with a pipette to mix.</h4>"+
+
"<h4>Gently blow it with a pipette to mix.</h4>"+
 
"<h4>b. First strand cDNA synthesis was performed under the following conditions:</h4>"+
 
"<h4>b. First strand cDNA synthesis was performed under the following conditions:</h4>"+
  
Line 338: Line 345:
  
 
"<h2>qPCR</h2>"+
 
"<h2>qPCR</h2>"+
−
"<h4>Use Vazyme ChamQ Universal SYBR® qPCR Master Mix</h4><br>"+
+
"<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>";
  
−
"<h2>Homologous recombination</h2>"+
 
−
"<h4>Use Vazyme ClonExpress II One Step Cloning kit</h4><br>";
 
  
 
var IF = "<h2>Sample preparation: </h2>"+
 
var IF = "<h2>Sample preparation: </h2>"+
−
"<h4>1.Grow cultured cells on chamber slides overnight, or add appropriate amount of cells to poly-L-lysine coated chamber slides and incubate at least 30 min at 37°C, at the time of fixation cells should be ~50% confluent. </h4>"+
+
"<h4>1.Grow cultured cells on chamber slides overnight. </h4>"+
 
"<h4>2.Rinse cells briefly in PBS. </h4>"+
 
"<h4>2.Rinse cells briefly in PBS. </h4>"+
−
"<h4>3.Fix cells by incubation with 4% Paraformaldehyde, in PBS for 15 min at room temperature. </h4>"+
+
"<h4>3.Fix cells by incubation with 4% Paraformaldehyde(Servicebio, China), in PBS for 15 min at room temperature. </h4>"+
 
"<h4>4.Rinse three times in PBS, 3 min each. </h4>"+
 
"<h4>4.Rinse three times in PBS, 3 min each. </h4>"+
 
"<h4>5.Add ice-cold acetone and incubate at -20°C for 10 min. </h4>"+
 
"<h4>5.Add ice-cold acetone and incubate at -20°C for 10 min. </h4>"+
Line 352: Line 357:
  
 
"<h2>Sample Blocking:</h2>"+
 
"<h2>Sample Blocking:</h2>"+
−
"<h4>Block samples in 5% normal serum from same species as secondary antibody in 1% BSA/0.2% Triton X-100/PBS for 1 h at room temperature, or overnight at 4°C.</h4><br>"+
+
"<h4>Block samples in 5% normal serum from same species as secondary antibody in 1% BSA/0.2% Triton X-100/PBS(Sinopharm Chmical Reagent, China) for 1 h at room temperature, or overnight at 4°C.</h4><br>"+
 
 
 
"<h2>Sample staining:</h2> "+
 
"<h2>Sample staining:</h2> "+
 
"<h4>7.Dilute the primary antibody to the recommended concentration/dilution in 1% BSA/0.05% Triton X-100/PBS. </h4>"+
 
"<h4>7.Dilute the primary antibody to the recommended concentration/dilution in 1% BSA/0.05% Triton X-100/PBS. </h4>"+
−
"<h4>8.Add 200 µl per well (8 wells) to the chamber slides and incubate 2 h at room temperature, or overnight at 4°C. </h4>"+
+
"<h4>8.Add 200 µl/well (8 wells) to the chamber slides and incubate 2 h at room temperature, or overnight at 4°C. </h4>"+
 
"<h4>9. Rinse three times in PBS, 3 min each.<h4>"+
 
"<h4>9. Rinse three times in PBS, 3 min each.<h4>"+
 
"<h4>10. Prepare fluorochrome-conjugated secondary antibody antibodies in 1% BSA/0.05% Triton X -100/PBS according to the recommended manufacturer specification data sheet and add 200 µl per well (8 wells) to the chamber slides. </h4>"+
 
"<h4>10. Prepare fluorochrome-conjugated secondary antibody antibodies in 1% BSA/0.05% Triton X -100/PBS according to the recommended manufacturer specification data sheet and add 200 µl per well (8 wells) to the chamber slides. </h4>"+
 
"<h4>11. Incubate the samples for 1 h at room temperature in dark. </h4>"+
 
"<h4>11. Incubate the samples for 1 h at room temperature in dark. </h4>"+
 
"<h4>12. Rinse three times in PBS, 3 min each. </h4>"+
 
"<h4>12. Rinse three times in PBS, 3 min each. </h4>"+
−
"<h4>13. Add DAPI solution in 1% BSA/0.05% Triton X -100/PBS, and incubate 15 min at room temperature.</h4>"+
+
"<h4>13. Add DAPI solution (Beyotime, China) in 1% BSA/0.05% Triton X -100/PBS, and incubate 15 min at room temperature.</h4>"+
 
"<h4>14. Rinse three times in PBS, 3 min each.</h4>"+
 
"<h4>14. Rinse three times in PBS, 3 min each.</h4>"+
 
"<h4>15. Coverslip with anti-fade mounting medium and seal slides with nail polish.</h4>"+
 
"<h4>15. Coverslip with anti-fade mounting medium and seal slides with nail polish.</h4>"+
−
"<h4>16. Take pictures under a fluorescence microscope</h4><br>";
+
"<h4>16. Take pictures under a fluorescence microscope</h4>"+
 +
        "<h4>17. Fluorescence microscopy.</h4>"
  
 
var WB = "<h2>Cell lysis to extract protein </h2>"+
 
var WB = "<h2>Cell lysis to extract protein </h2>"+
Line 370: Line 376:
 
 
 
"<h2>Adherent cells:</h2>"+
 
"<h2>Adherent cells:</h2>"+
−
"<h4>1.Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS. (Tip: Keep tissue culture dish on ice throughout).</h4>"+
+
"<h4>(1) Wash cells by adding cold phosphate buffered saline (PBS) and shake it gently. Discard the PBS. (Tip: Keep tissue culture dish on ice throughout the operation).</h4>"+
−
"<h4>2.Add PBS and use a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes.</h4>"+
+
"<h4>(2) Add PBS and use a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes.</h4>"+
−
"<h4>3.Centrifuge at 1500 RPM for 5 minutes and discard the supernatant.</h4>"+
+
"<h4>(3) Centrifuge at 1500 RPM for 5 minutes and discard the supernatant.</h4>"+
−
"<h4>4.Add 180 μL of ice cold cell lysis buffer with 20 μL fresh protease inhibitor cocktail. (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail).</h4>"+
+
"<h4>(4) Add 180 μL of ice cold cell lysis buffer with 20 μL of fresh protease inhibitor cocktail (Beyotime, China). (Tip: If the protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail).</h4>"+
−
"<h4>5.Incubate for 30 minutes on ice, and then clarify the lysate by spinning for 10 minutes at 12,000 RPM, at 4°C.</h4>"+
+
"<h4>(5) Incubate for 30 minutes on ice, and then clarify the lysate by centrifugation for 10 minutes at 12,000 RPM, at 4°C.</h4>"+
−
"<h4>6.Transfer supernatant (or protein mix) to a fresh tube and store on ice or frozen at -20°C or -80°C.</h4>"+
+
"<h4>(6) Transfer the supernatant (or protein mix) to a fresh tube and store it on ice or frozen at -20°C or -80°C.</h4>"+
−
"<h4>7.Measure the concentration of protein using a spectrophotometer.</h4><br>"+
+
"<h4>(7) Measure the concentration of protein using a spectrophotometer.</h4><br>"+
 
 
 
"<h2>Sample preparation</h2>"+
 
"<h2>Sample preparation</h2>"+
−
"<h4>1.</h4>"+
+
"<h4>(1)        Determine the volume of protein extract to ensure that there will be 50 μg in each well.</h4>"+
 
         "<center><img style=\"width:60% !important;\" src=\"https://static.igem.org/mediawiki/2018/6/6b/T--CPU_CHINA--wb-1.png\"></center>"+
 
         "<center><img style=\"width:60% !important;\" src=\"https://static.igem.org/mediawiki/2018/6/6b/T--CPU_CHINA--wb-1.png\"></center>"+
 
"<h4>determine the volume of protein extract to ensure 50 μg in each well.</h4>"+
 
"<h4>determine the volume of protein extract to ensure 50 μg in each well.</h4>"+
−
"<h4>2.Add 5 μL sample buffer to the sample, and make the volume in each lane equalized using double distilled H2O (dd H2O). Mix well. (Tip: Total volume of 15 μL per lane is suggested).</h4>"+
+
"<h4>(2) Add 5 μL of sample buffer into the sample and make the volume in each lane equal using double distilled H2O (ddH2O). Mix them well. (Tip: Total volume of 15 μL per lane is suggested).</h4>"+
−
"<h4>3.Heat the samples with dry plate for 5 minutes at 100°C.</h4><br>"+
+
"<h4>(3) Heat the samples with dry plate for 5 minutes at 100°C.</h4><br>"+
 
 
 
"<h2>Gel preparation</h2>"+
 
"<h2>Gel preparation</h2>"+
 
         "<center><img style=\"width:60% !important;\" src=\"https://static.igem.org/mediawiki/2018/3/31/T--CPU_CHINA--wb-2.png\"></center>"+
 
         "<center><img style=\"width:60% !important;\" src=\"https://static.igem.org/mediawiki/2018/3/31/T--CPU_CHINA--wb-2.png\"></center>"+
−
"<h4>1.After preparing the 10% stacking gel solution, assemble the rack for gel solidification. (Tip: 10% AP and TEMED solidify the solution; therefore, both gels can be prepared at the same time, if the abovementioned reagents are not added until the end).</h4>"+
+
"<h4>(1) After preparing the 10% stacking gel solution, assemble the rack for gel solidification. (Tip: 10% AP and TEMED solidify the solution; therefore, before addition of them, both gel solutions simultaneously can be prepared).</h4>"+
−
"<h4>2.Add stacking gel solution carefully until the level is equal to the green bar holding the glass plates. Add H2O to the top. Wait for 15–30 minutes until the gel turning solidified. (Tip: Using a suction pipette can make the process of adding the gel to the glass plate easier).</h4>"+
+
"<h4>(2) Add stacking gel solution carefully until the level is equal to the green bar holding the glass plates. Add H2O onto the top. Wait for 15–30 minutes until the gel solidifies. (Tip: A suction pipette can help make the process of adding the gel to the glass plate easier).</h4>"+
−
"<h4>3.Overlay the stacking gel with the separating gel, after removing the water. (Tip: It is better to tilt the apparatus and use a paper towel to remove the water).</h4>"+
+
"<h4>(3) Overlay the stacking gel with the separating gel, after removing the water. (Tip: It is better to tilt the apparatus and use a paper towel to remove the water).</h4>"+
−
"<h4>4.Insert the comb, ensuring that there are no air bubbles.</h4>"+
+
"<h4>(4) Insert the comb, ensuring that there are no air bubbles.</h4>"+
−
"<h4>5.Wait until the gel is solidified. (Tip: Solidification can be easily checked by leaving some gel solution in a tube).</h4><br>"+
+
"<h4>(5) Wait until the gel solidifies. (Tip: Solidification can be easily checked by leaving some gel solution in a tube).</h4><br>"+
 
 
 
"<h2>Electrophoresis</h2>"+
 
"<h2>Electrophoresis</h2>"+
−
"<h4>1.Pour the running buffer into the electrophorator</h4>"+
+
"<h4>(1).Pour the running buffer into the electrophorator</h4>"+
−
"<h4>2.Place gel inside the electrophorator and connect to a power supply. (Tip: When connecting to the power source always connect red to red, and black to black).</h4>"+
+
"<h4>(2).Place gel inside the electrophorator and connect to a power supply. (Tip: When connecting to the power source always connect red to red, and black to black).</h4>"+
−
"<h4>3.Make sure buffer covers the gel completely, and remove the comb carefully.</h4>"+
+
"<h4>(3).Make sure buffer covers the gel completely, and remove the comb carefully.</h4>"+
−
"<h4>4.Load marker (6 μL) followed by samples (15 μL) in to each well</h4>"+
+
"<h4>(4).Load marker (6 μL) followed by samples (15 μL) in to each well</h4>"+
−
"<h4>5.Run the gel with low voltage (60 V) for separating gel; use higher voltage (140 V) for stacking gel.</h4>"+
+
"<h4>(5).Run the gel with low voltage (60 V) for separating gel; use higher voltage (140 V) for stacking gel.</h4>"+
−
"<h4>6.Run the gel for approximately an hour, or until the dye front runs off the bottom of the gel.</h4><br>"+
+
"<h4>(6).Run the gel for approximately an hour, or until the dye front runs off the bottom of the gel.</h4><br>"+
 
 
 
"<h2>Electrotransfer</h2>"+
 
"<h2>Electrotransfer</h2>"+
−
"<h4>1.Cut 6 filter sheets to fit the measurement of the gel, and one polyvinylidene fluoride (PDVF) membrane with the same dimensions.</h4>"+
+
"<h4>(1) Cut 6 pieces of filter sheet and one polyvinylidene fluoride (PDVF) membrane (Millipore, US) to fit the measurements of the gel.</h4>"+
−
"<h4>2.Wet the sponge and filter paper in transfer buffer, and wet the PDVF membrane in methanol.</h4>"+
+
"<h4>(2) Wet the sponge and filter paper in transfer buffer and wet the PDVF membrane in methanol.</h4>"+
−
"<h4>3.Separate glass plates and retrieve the gel.</h4>"+
+
"<h4>(3) Separate the glass plates and retrieve the gel.</h4>"+
−
"<h4>4.Create a transfer sandwich as follows:</h4>"+
+
"<h4>(4) Create a transfer sandwich as follow:</h4>"+
 
"<h4>Sponge</h4>"+
 
"<h4>Sponge</h4>"+
 
"<h4>3 Filter Papers</h4>"+
 
"<h4>3 Filter Papers</h4>"+
Line 411: Line 417:
 
"<h4>PVDF membrane</h4>"+
 
"<h4>PVDF membrane</h4>"+
 
"<h4>3 Filter Papers</h4>"+
 
"<h4>3 Filter Papers</h4>"+
−
"<h4>(Tip: Ensure there are no air bubbles between the gel and PVDF membrane, and squeeze out extra liquid).</h4>"+
+
"<h4>(Tip: Ensure that there are no air bubbles between the gel and PVDF membrane, and squeeze out extra liquid).</h4>"+
−
"<h4>5.Place electrodes on top of the sandwich, ensuring that the PVDF membrane is between the gel and a positive electrode. Parameter setting of the transmembrane: A=[Area of Gel*3/4]/1000, constant current mode.</h4>"+
+
"<h4>(5).Place electrodes on top of the sandwich, ensuring that the PVDF membrane is between the gel and a positive electrode. Parameter setting of the transmembrane: A=[Area of Gel*3/4]/1000, constant current mode.</h4>"+
−
"<h4>6.Transfer for 7 minutes. </h4>"+
+
"<h4>(6).Transfer for 7 minutes. </h4>"+
−
"<h4>7.Wash the membrane with ddH2O for 5min. Do this 3 times. (Tip: Shaking the membrane slowly.)</h4><br>"+
+
"<h4>(7) Wash the membrane with ddH2O for 5min. Do this for 3 times. (Tip: Shake the membrane slowly.)</h4><br>"+
 
 
 
"<h2>Blocking and antibody incubation</h2>"+
 
"<h2>Blocking and antibody incubation</h2>"+
−
"<h4>1.Block the membrane with 3% bovine serum albumin ( BSA) at 37℃，350r/min shaking, for 2 hour. </h4>"+
+
"<h4>(1) Block the membrane with 3% bovine serum albumin (BSA) (Biofoxx, Germany) at 37℃，350r/min shaking, for 2 hours. </h4>"+
−
"<h4>2.Wash the PVDF membrane with TBST Buffer for 10min. Do this 3 times. (Tip: Shaking the membrane slowly.)</h4>"+
+
"<h4>(2) Wash the PVDF membrane with TBST Buffer for 10min. Do this Repeat for 3 times. (Tip: Shake the membrane slowly.)</h4>"+
−
"<h4>3.Add primary antibody in 5% bovine serum albumin ( BSA) and incubate overnight in 4°C on a shaker.</h4>"+
+
"<h4>(3) Add primary antibody in 5% bovine serum albumin (BSA) and incubate overnight in 4°C on a shaker.</h4>"+
−
"<h4>4.Wash the membrane with TBST for 5 minutes. Do this 3 times. (Tip: All washing and antibody incubation steps should be done on a shaker at room temperature to ensure even agitation).</h4>"+
+
"<h4>(4) Wash the membrane with TBST for 5 minutes. Do this Repeat for 3 times. (Tip: All washing and antibody incubation steps should be done on a shaker at room temperature to ensure even agitation).</h4>"+
−
"<h4>5.Add secondary antibody in 5% skim milk in TBST, and incubate for 1 hour.</h4>"+
+
"<h4>(5) Add secondary antibody in 5% skim milk in TBST and incubate for 1 hour.</h4>"+
−
"<h4>6.Wash the membrane with TBST for 5 minutes. Do this 3 times</h4>"+
+
"<h4>(6) Wash the membrane with TBST for 5 minutes. Do this for 3 times.</h4>"+
−
"<h4>7.Prepare ECL mix (following the proportion of solution A and B provided by the manufacturer). Incubate the membrane for 1–2 minutes. (Tip: Use a 1000 μL pipette to ensure that ECL covers the top and bottom of the membrane).</h4>"+
+
"<h4>(7) Prepare ECL mix (Millipore, US) (following the proportion of solution A and B provided by the manufacturer). Incubate the membrane for 1–2 minutes. (Tip: Use a 1000 μL pipette to ensure that ECL covers the top and bottom of the membrane).</h4>"+
−
"<h4>8.isualize the result in the dark room. (Tip: If the background is too strong, reduce exposure time).</h4><br>"+
+
"<h4>(8) Visualize the result in a dark room. (Tip: If the background is too strong, reduce exposure time).</h4><br>";
−
+
−
"<h2>Recipe</h2>"+
+
−
"<h4>1.Dissolve the following in 800 ml of distilled H2O</h4>"+
+
−
"<h4>● 8.8 g of NaCl</h4>"+
+
−
"<h4>● 0.2g of KCl</h4>"+
+
−
"<h4>● 3g of Tris base</h4>"+
+
−
"<h4>2.Add 500ul of Tween-20</h4>"+
+
−
"<h4>3.Adjust the pH to 7.4</h4>"+
+
−
"<h4>4.Add distilled H2O to 1L</h4>"+
+
−
"<h4>5.Sterilize by filtration or autoclaving</h4><br>";
+
  
 
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>"+
−
"<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>T--CPU_CHINA--hp-aiyou.webm</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>";
+
 
 
 
 
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var x=query.split('=');
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 +
  if(getQueryVariable()=='promoters'){showExperiment(5);}
 +
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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.