Team:NJU China/Wet lab
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<li><a href="https://2013.igem.org/Team:NJU_China/Project">Project</a> | <li><a href="https://2013.igem.org/Team:NJU_China/Project">Project</a> | ||
<ul> | <ul> | ||
- | <li><a href="https://2013.igem.org/Team:NJU_China/Project">Overview</a></li> | + | <li><a href="http://https://2013.igem.org/Team:NJU_China/Project#Overview">Overview</a></li> |
- | <li><a href="https://2013.igem.org/Team:NJU_China/Project">Chassis</a></li> | + | <li><a href="https://2013.igem.org/Team:NJU_China/Project#Chassis">Chassis</a></li> |
- | <li><a href="https://2013.igem.org/Team:NJU_China/Project">Targeting Module</a></li> | + | <li><a href="https://2013.igem.org/Team:NJU_China/Project#Targeting Module">Targeting Module</a></li> |
- | <li><a href="https://2013.igem.org/Team:NJU_China/Project">Killing Module</a></li> | + | <li><a href="https://2013.igem.org/Team:NJU_China/Project#Killing Module">Killing Module</a></li> |
- | <li><a href="https://2013.igem.org/Team:NJU_China/Project">Achievement</a></li> | + | <li><a href="https://2013.igem.org/Team:NJU_China/Project#Achievement">Achievement</a></li> |
</ul> | </ul> | ||
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<li><a href="https://2013.igem.org/Team:NJU_China/Human Practice">Human Practice</a> | <li><a href="https://2013.igem.org/Team:NJU_China/Human Practice">Human Practice</a> | ||
<ul> | <ul> | ||
- | <li><a href="https://2013.igem.org/Team:NJU_China/ | + | <li><a href="http://https://2013.igem.org/Team:NJU_China/Project#Free debate">Free debate</a></li> |
- | <li><a href="https://2013.igem.org/Team:NJU_China/ | + | <li><a href="https://2013.igem.org/Team:NJU_China/Project#Reach the unreached">Reach the unreached</a></li> |
- | <li><a href="https://2013.igem.org/Team:NJU_China/ | + | <li><a href="https://2013.igem.org/Team:NJU_China/Project#Workshop">Workshop</a></li> |
- | <li><a href="https://2013.igem.org/Team:NJU_China/ | + | <li><a href="https://2013.igem.org/Team:NJU_China/Project#Collaboration">Collaboration</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2013.igem.org/Team:NJU_China/Wet lab">Wet lab</a> | <li><a href="https://2013.igem.org/Team:NJU_China/Wet lab">Wet lab</a> | ||
<ul> | <ul> | ||
- | <li><a href="https://2013.igem.org/Team:NJU_China/Protocol">Protocol</a></li> | + | <li><a href="http://https://2013.igem.org/Team:NJU_China/Project#Protocol">Protocol</a></li> |
<li><a href="https://2013.igem.org/Team:NJU_China/Notebook">Lab notes</a></li> | <li><a href="https://2013.igem.org/Team:NJU_China/Notebook">Lab notes</a></li> | ||
- | <li><a href="https://2013.igem.org/Team:NJU_China/ | + | <li><a href="https://2013.igem.org/Team:NJU_China/Data_Page">Data Page</a></li> |
<li><a href="https://2013.igem.org/Team:NJU_China/Parts">Parts</a></li> | <li><a href="https://2013.igem.org/Team:NJU_China/Parts">Parts</a></li> | ||
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<li><a href="https://igem.org/2013_Judging_Form?id=1180">Judging criteria</a></li> | <li><a href="https://igem.org/2013_Judging_Form?id=1180">Judging criteria</a></li> | ||
- | <li><a href="https://2013.igem.org/Team:NJU_China/ | + | <li><a href="https://2013.igem.org/Team:NJU_China/Extras">Attribution</a></li> |
<li><a href="https://2013.igem.org/Team:NJU_China/Acknowledgement">Acknowledgement</a></li> | <li><a href="https://2013.igem.org/Team:NJU_China/Acknowledgement">Acknowledgement</a></li> | ||
</ul> | </ul> | ||
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- | <!--End NavBar--> | + | <!--End NavBar--> |
- | <h2 class="ss-subtitle">Notebook</h2> | + | <h2 class="ss-subtitle">Notebook</h2> |
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<a href="#March">Mar</a> | <a href="#March">Mar</a> | ||
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<a href="#August">Aug</a> | <a href="#August">Aug</a> | ||
<a href="#September">Sep</a> | <a href="#September">Sep</a> | ||
+ | <a href="#October">Oct</a> | ||
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<h3> | <h3> | ||
<a>WEEK 8</a> | <a>WEEK 8</a> | ||
- | <span> | + | <span>19th-24th, June</span> |
+ | </h3> | ||
+ | </div> | ||
+ | <div class="ss-right"> | ||
+ | <h3> | ||
+ | <a>siRNA screening (failed)</a> | ||
+ | <span> | ||
+ | 8th: We cultured Hep G2 cells in 2 12-well plates for 24-hour cell collection and 48-hour cell collection.</br> | ||
+ | 9th: We transfected cells with empty Lipo, HbsAg plasmids, HbsAg plasmids and 467 siRNA plasmids, HbsAg plasmids and 516 siRNA plasmids, respectively.</br> | ||
+ | 10th: We collected cells 24 hours after transfection, and preserved them in Trizol.</br> | ||
+ | 11th: We collected 48 hours after transfection, and then extracted RNA from 2 groups of cells (24 hours and 48 hours).</br> | ||
+ | 12th: We did RT-PCR and qPCR with all RNA samples.</br> | ||
+ | 13th: We analyzed the data.</br> | ||
+ | </span> | ||
+ | </h3> | ||
+ | </div> | ||
+ | </div> | ||
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+ | <div class="ss-row"> | ||
+ | <div class="ss-left"> | ||
+ | <h2 id="July">July</h2> | ||
+ | </div> | ||
+ | <div class="ss-right"> | ||
+ | <h2>2013</h2> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="ss-row ss-medium"> | ||
+ | <div class="ss-left"> | ||
+ | <h3> | ||
+ | <a>WEEK 8</a> | ||
+ | <span>19th-24th, June</span> | ||
</h3> | </h3> | ||
</div> | </div> | ||
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26th: We analyzed data and found that the standard curve cannot be used.</br> | 26th: We analyzed data and found that the standard curve cannot be used.</br> | ||
27th: We redid the Q-PCR and analyzed data and this time we succeeded</br> | 27th: We redid the Q-PCR and analyzed data and this time we succeeded</br> | ||
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</span> | </span> | ||
<a>Examination whether RVG-lamp2b exosomes will target to dendritic cells or not (failed)</a> | <a>Examination whether RVG-lamp2b exosomes will target to dendritic cells or not (failed)</a> | ||
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<h3> | <h3> | ||
<a>WEEK 16-17</a> | <a>WEEK 16-17</a> | ||
- | <span>2th- | + | <span>2th-11th, September</span> |
</h3> | </h3> | ||
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4th: We redid RT-PCR and Q-PCR on 3th September.</br> | 4th: We redid RT-PCR and Q-PCR on 3th September.</br> | ||
</span> | </span> | ||
- | <a> | + | <a>Exosomes collection</a> |
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4th: We transfected 293T cells with 467 plasmids.</br> | 4th: We transfected 293T cells with 467 plasmids.</br> | ||
5th: We soaked centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).</br> | 5th: We soaked centrifuge tubes which then be used in ultracentrifugation overnight in DEPC solution(1:1000).</br> | ||
6th: We collected culture medium and separated exosomes by ultracentrifugation and stored the exosome solution at 4℃.</br> | 6th: We collected culture medium and separated exosomes by ultracentrifugation and stored the exosome solution at 4℃.</br> | ||
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</span> | </span> | ||
<a>Construction of standardized plasmid</a> | <a>Construction of standardized plasmid</a> | ||
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11th: We got result of sequencing from GenScript. We constructed standardized 467-plasmid successfully.</br> | 11th: We got result of sequencing from GenScript. We constructed standardized 467-plasmid successfully.</br> | ||
</span> | </span> | ||
- | <a> | + | </h3> |
- | <span> | + | </div> |
- | + | </div> | |
+ | |||
+ | |||
+ | <div class="ss-row"> | ||
+ | <div class="ss-left"> | ||
+ | <h2 id="October">October</h2> | ||
+ | </div> | ||
+ | <div class="ss-right"> | ||
+ | <h2>2013</h2> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="ss-row ss-medium"> | ||
+ | <div class="ss-left"> | ||
+ | <h3> | ||
+ | <a>Week 1</a> | ||
+ | <span>12th-19th, October</span> | ||
+ | </h3> | ||
+ | </div> | ||
+ | <div class="ss-right"> | ||
+ | <h3> | ||
<span> | <span> | ||
+ | 12th : Subcultured 293T cells to six 225 cm2 flasks and three 75 cm2 flasks .</br> | ||
+ | 14th:Transferred 293T cells with plasmids and microRNA.For the six 225cm2 flasks , three groups : lipo*2, mirco162 RNA*2, micro162 RNA + GP120 plasmid(targeting to T cell) *26 hours After transfecting, Changed the culture mediaSubcultured 293T cells.</br> | ||
+ | 15th : Soaked centrifuge tubes which will be used in ultracentrifugation over night in ddH2O</br> | ||
+ | 16th: Collected 36-hour~48-hour culture containing exosomes.(The cells transfected with micro162 were contaminated by bacterium). Did pre-centrifuge(1500g:10min; 10000g: 30min). Separated exosomes by ultracentrifugation. Examine protein concentration of exosomes.Subcultured 293T cells.</br> | ||
+ | 17th: Separated T cells from the spleen of mouse, cultured the cells in 24-well plate, Each well contained 10^5 cells. cocultured empty exosomes and mirco162+GP120 exosomes with T cell respectively, each well added 10ug exosomes.Cultured HepG2 cells to 15 wells in 96-well plate, and did 4 plates. </br> | ||
+ | 18th : collected T cell in 12-well plate, separated CD4+ cells and CD4- cells using CD4+ T cell isolation Kit ,and started to extract RNA from the cells respectively, and then RT-Q-PCR.Subcultured 293T cells.HepG2 cells were contaminated by bacterium.</br> | ||
+ | 19th : Analyzed data.</br> | ||
+ | </span> | ||
</h3> | </h3> | ||
</div> | </div> | ||
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<h3> | <h3> | ||
- | <a> | + | <a>Week 2</a> |
- | <span> | + | <span>20th-26th, October</span> |
</h3> | </h3> | ||
</div> | </div> | ||
<div class="ss-right"> | <div class="ss-right"> | ||
<h3> | <h3> | ||
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<span> | <span> | ||
+ | 20th : Subcultured 293T cells</br> | ||
+ | 22th : transferred 293T cells with plasmids and microRNA(before transfecting, collected the medium for empty exosomes).For twelve 225 cm2 flasks, micro162 RNA*2, micro162 RNA+ GP120 plasmid *2, GFP inhibit siRNA *8; For twenty four 75 cm2 flasks, GFP inhibit siRNA + pres1 plasmid;cultured HepG2 cells to 15 wells in 96-well plate, and did 5 plates.</br> | ||
+ | 23th : Separated T cells from the spleen of mouse, cultured the cells in 24-well plate, Each well contained 3.2 *10^5 cells. (0 hours after adding exosomes into HepG2 cells) collected the medium of one plate with HepG2 cells for elisa. And Added 10ul CCK reagent and put the plate in incubator for 3hours, then tested it’s OD value. Add exosomes into HepG2 cells in each plate of the remaining four: PBS 10ul each well *5; empty exosomes 10ul(3ug) each well *5; micro162+GP120 exosomes 10ul(3ug) each well *5; Soaked centrifuge tubes which will be used in ultracentrifugation over night in ddH2O</br> | ||
+ | 24th : Collected 36-hour~48-hour culture containing exosomes.Cocultured empty exosomes, mirco162 exosomes and mirco162+GP120 exosomes with T cell respectively, each well added 10ug exosomes.12 hours after adding exosomes into HepG2 cells, collected the medium for elisa. Added 10ul CCK reagent and put the plate in incubator for 3hours, then tested it’s OD value. For 24/36/48 hours after adding exosomes into HepG2 cells, did the same experiments as 0/12hours.</br> | ||
+ | 25th : Collected T cell in 12-well plate, separated CD4+ cells and CD4- cells using CD4+ T cell isolation Kit ,and started to extract RNA from the cells respectively, and then RT-Q-PCR.Got the OD value of 0/12/24/36/48 hours after adding exosomes in HepG2 cells. Injected three GFP mice with empty exosomes , exosomes containing GFP inhibit siRNA, and pres1-exosomes with GFP inhibit siRNA respectively twice. For the first time at 9 am and the second time at 9 pm.</br> | ||
+ | 26th : Analyzed data </br> | ||
+ | Killed the three GFP mice and took out their liver respectively at 9 am. </br> | ||
+ | Did a frozen section of the hepatic tissue.</br> | ||
+ | Observed the slice of the tissue with the fluorescence microscope and took pictures.</br> | ||
+ | Using the Human IL-6 Elisa kit, Human IL-8 Elisa kit and Human TNF-α Elisa kit to test OD450 value of the medium we collected 0/12/24/36/48 hours after adding exosomes in HepG2 cells. </br> | ||
+ | Analyzed data.</br> | ||
+ | </span> | ||
</h3> | </h3> | ||
</div> | </div> | ||
</div> | </div> | ||
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+ | </div> | ||
+ | </div> | ||
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Latest revision as of 01:22, 29 October 2013