Team:MIT mge
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- | <a href="https://2010.igem.org/Team:MIT_bconst"> | + | <dl id="nav"> |
- | <a href="https://2010.igem.org/Team:MIT_bexp">Bacterial | + | <dt><b>Bacterial Protocol</b></dt> |
- | + | <dd> | |
- | <a href="https://2010.igem.org/Team:MIT_mmethods"> | + | <ul> |
- | + | <li><a href="https://2010.igem.org/Team:MIT_bconst">Biobrick Construction</a></li> | |
- | + | <li><a href="https://2010.igem.org/Team:MIT_bexp">Bacterial Experiments</a></li> | |
- | <a href="https://2010.igem.org/Team: | + | </ul> |
- | <a href="https://2010.igem.org/Team: | + | </dd> |
+ | <dt><b>Mammalian Protocol</b></dt> | ||
+ | |||
+ | <dd> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:MIT_mmethods">Microfluidics</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:MIT_gateway">Gateway Cloning</a></li> | ||
+ | |||
+ | </ul> | ||
+ | </dd> | ||
+ | <dt><b>Phage Protocol</b></dt> | ||
+ | |||
+ | <dd> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:MIT_phageprot">Basic Protocol</a></li> | ||
+ | |||
+ | </ul> | ||
+ | </dd> | ||
+ | |||
+ | </dl> | ||
</div> | </div> | ||
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- | <table width= | + | <div id="unique" style="padding:0px; font-size: 14px; border: 1px solid black; margin:0px; background-color:transparent;"> |
- | <tr><td><br>The Mammalian team | + | <table width=650px style="background-color: white; margin-top:5px; padding: 10px;"> |
+ | <tr><td><div class="bodybaby">mammalian genetic engineering protocol</div></td> | ||
+ | <tr><td><br>The Mammalian team created lines of cells using lentiviruses for transfection.<br><br> | ||
<div class="outline"> | <div class="outline"> | ||
- | <a href="# | + | <a href="#cpt">1 Calcium Phosphate Transfection</a><br> |
- | <a href="# | + | <a href="#before">1.1 Preparation</a><br> |
- | <a href="# | + | <a href="#prot">1.2 Protocol</a><br> |
- | + | <a href="#filter">2 Filter and Concentrate Virus</a><br> | |
- | + | <a href="#proto">2.1 Protocol</a><br> | |
- | <a href="# | + | <a href="#materials">3 Materials Needed</a><br> |
- | + | <a href="#hbss">3.1 HBSS Buffer</a><br> | |
- | + | <a href="#cacl">3.2 Calcium Chloride Solution</a><br> | |
- | <a href="# | + | <a href="#freeze">3.3 Freezing medium</a><br> |
- | + | <a href="#dmem">3.4 Supplemented DMEM</a><br> | |
- | + | ||
- | <a href="# | + | |
- | <a href="# | + | |
- | <a href="# | + | |
</div></td><tr><td><br> | </div></td><tr><td><br> | ||
- | <div class="bodybaby" id=" | + | <div class="bodybaby" id="cpt">Calcium Phosphate Transfection</div><br> |
- | <b class="bolded" id=" | + | <b class="bolded" id="before">Beforehand:</b><br> |
<ul id="procedure"> | <ul id="procedure"> | ||
<li>Combine CaCL2 and dilute Na2PO4 to get CaPO4 with DNA in it</li> | <li>Combine CaCL2 and dilute Na2PO4 to get CaPO4 with DNA in it</li> | ||
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<ul><li>No cells (see precipitate, like fine snow on cells)</li><li>Just carrier DNA (any plasmid)</li></ul> | <ul><li>No cells (see precipitate, like fine snow on cells)</li><li>Just carrier DNA (any plasmid)</li></ul> | ||
<li></li> | <li></li> | ||
+ | </ul><br> | ||
+ | <b class="bolded" id="prot">Protocol</b><br> | ||
+ | <ul id="procedure"> | ||
+ | <li>1. Mix DNA (different plasmids in right amounts) with CaCl2</li> | ||
+ | <li>2. Quickly vortex in 14 mL tube</li> | ||
+ | <li>3. Add water to ~1.5 mL total volume</li> | ||
+ | <li>4. Must saturate water with air - vortex without cap for 20-30 sec</li> | ||
+ | <li>5. Make 1.5 mL HBSS aliquot</li> | ||
+ | <li>6. For each tfxn, we now have 1 tube HBSS and 1 tube DNA+CaCl2+H2O in 14 mL tubes</li> | ||
+ | <li>7. Add DNA solution dropwise to HBSS - 10-20 sec total - while vortexing solution. Use 2mL pipette for adding</li> | ||
+ | <li>8. Immediately suck up into 5mL pipette and stop reaction by loading onto cells (spread by pipetting onto medium, no need to shake</li> | ||
+ | <li>9. Check precipitation under microscope (small black dots between cells)</li> | ||
+ | <li>10. Put cells back into incubator</li> | ||
+ | <li>11. Next morning, change medium</li> | ||
+ | <li>12. 1 and again 2 days after: Harvest 20mL (i.e. all the medium) to filter and centrifuge to obtain virus.</li> | ||
</ul> | </ul> | ||
<br> | <br> | ||
- | <b class="bolded" id=" | + | <div class="bodybaby" id="filter">Filter and Concentrate Virus</div><br> |
+ | <b class="bolded" id="proto">Protocol</b><br> | ||
+ | <ul id="procedure"> | ||
+ | <li>1. Start with 20 + 20 mL culture supernatant</li> | ||
+ | <li>2. Ultracentrifuge tubes (kept on shelf in tissue culture room) may contain residual bleach</li> | ||
+ | <ul><li>Wash 2x with 70% EtOH</li><li>Wash 1x with medium</li></ul> | ||
+ | <li>3. Filter through 0.45 um (0.2 um can be used) Corning vacuum filter into 50 mL tube into ultracentrifuge tube</li> | ||
+ | <li>4. Everything that contacts the virus including tips and plates must be bleached</li> | ||
+ | <li>5. Spray filters with 10% bleach</li> | ||
+ | <li>6. After filtered supernatant, add to each tube 4-5 mL sterile 20% w/v sucrose in PBS solution</li> | ||
+ | <ul><li>Purpose of sucrose is to remove excess VSVG protein and dampen impact of particles</li><li>Phases will mix a little</li></ul> | ||
+ | <li>7. Balance weight with PBS to within <5mg </li> | ||
+ | <li>8. Ultracentrifugation</li> | ||
+ | <ul><li>Speed: 45,000 RCF (g)</li><li>Rotof: JA 25.50 (max 8 tubes)</li><li>Time: 3h</li><li>Accel: slow</li><li>Decel: OFF</li><li>Temp: 4°C</li></ul> | ||
+ | <li>9. When finished, cannot wait too long or pellet detaches</li> | ||
+ | <li>10. Mark pellet, then aspirate supernatant</li> | ||
+ | <li>11. Resuspend in 100uL PBS (or HBSS) for total of ~150 uL</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <div class="bodybaby" id="materials">Materials</div><br> | ||
+ | <b class="bolded" id="hbss">HBSS Buffer (500 mL)</b><br> | ||
+ | <ul id="procedure"> | ||
+ | <li><ul><li>8.0g NaCl</li> | ||
+ | <li>0.37g KCl</li> | ||
+ | <li>106.5mg Na2HPO4 (anhydrous, 201.1 mg if 7x H2O)</li> | ||
+ | <li>1.0g dextrose</li> | ||
+ | <li>5.0g Hepes</li></ul></li> | ||
+ | <li>Add all components into a beaker and add 400 mL of Nanopure/bidistilled water. Dissolve and adjust pH exactly to 7.06 (wait 20 min. to be sure that the pH won't change anymore).</li> | ||
+ | <li>Filter through a 0.2 um filter, aliquot into 15 mL plastic tubes (12mL/tube). Can be kept at -20 for a few months.</li></ul><br> | ||
+ | <b class="bolded" id="cacl">Calcium Chloride Solution (50mL)</b> | ||
+ | <ul id="procedure"> | ||
+ | <li>5M CaCl2 (18.4g CaCl2*2H2O in 50mL)</li> | ||
+ | <li>Keep at +4°C</li></ul><br> | ||
+ | <b class="bolded" id="freeze">Freezing medium</b> | ||
+ | <ul id="procedure"> | ||
+ | <li>12mL FCS</li> | ||
+ | <li>15mL DMEM (no antibiotics, directly as supplied)</li> | ||
+ | <li>3mL DMSO</li> | ||
+ | <li>TOTAL: 30mL</li></ul><br> | ||
+ | <b class="bolded" id="dmem">Supplemented DMEM</b> | ||
<ul id="procedure"> | <ul id="procedure"> | ||
- | <li> | + | <li>450mL DMEM +Glucose +glutamine</li> |
- | <li> | + | <li>50mL FBS</li> |
- | < | + | <li>5mL Pen/Strep</li> |
+ | <li>0.5mL Fungin</li> | ||
+ | <li>TOTAL: 500mL</li></ul> | ||
</td> | </td> |
Latest revision as of 16:54, 27 October 2010
mammalian genetic engineering protocol |
The Mammalian team created lines of cells using lentiviruses for transfection. |
Calcium Phosphate Transfection Beforehand:
Protocol
Filter and Concentrate Virus Protocol
Materials HBSS Buffer (500 mL)
Calcium Chloride Solution (50mL)
Freezing medium
Supplemented DMEM
|