Team:MIT mge

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<div class="bodybaby" style="font-size: 12px;"><a color=black href="https://2010.igem.org/Team:MIT_mm">Methods</a></div><br>
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<div style="width:250px; margin: 10px; position: relative; top: -4px; left:-11px; display: block; float:right; padding: 7px; background-color: white;">
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<a href="https://2010.igem.org/Team:MIT_bconst">Bacterial Construction Protocol</a><br>
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<dl id="nav">
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<a href="https://2010.igem.org/Team:MIT_bexp">Bacterial Experimental Protocol</a><br>
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<dt><b>Bacterial Protocol</b></dt>
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Phage western blot<br>
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<dd>
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<a href="https://2010.igem.org/Team:MIT_mmethods">Mammalian Protocol</a><br>
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<ul>
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Microfluidic stress<br>
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<li><a href="https://2010.igem.org/Team:MIT_bconst">Biobrick Construction</a></li>
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<br><div class="bodybaby" style="font-size: 12px;">Mammalian Protocol</div><br>
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<li><a href="https://2010.igem.org/Team:MIT_bexp">Bacterial Experiments</a></li>
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<a href="https://2010.igem.org/Team:MIT_mmethods">Microfluidics Protocol</a><br>
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</ul>
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<a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering Protocol</a><br>
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</dd>
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<dt><b>Mammalian Protocol</b></dt>
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<dd>
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<ul>
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<li><a href="https://2010.igem.org/Team:MIT_mmethods">Microfluidics</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering</a></li>
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<li><a href="https://2010.igem.org/Team:MIT_gateway">Gateway Cloning</a></li>
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</ul>
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</dd>
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<dt><b>Phage Protocol</b></dt>
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<dd>
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<ul>
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<li><a href="https://2010.igem.org/Team:MIT_phageprot">Basic Protocol</a></li>
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</ul>
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</dd>
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</dl>
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<div id="unique" style="padding:5px; font-size: 14px; border: 1px solid black; margin:5px;">
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<table width=70%><tr><td><div class="bodybaby">mammalian microfluidic protocol</div></td>
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<div id="unique" style="padding:0px; font-size: 14px; border: 1px solid black; margin:0px; background-color:transparent;">
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<tr><td><br>The Mammalian team focused on creating a pressure-sensitive promoter and creating a standard protocol for mammalian genes.<br><br>
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<table width=650px style="background-color: white; margin-top:5px; padding: 10px;">
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<tr><td><div class="bodybaby">mammalian genetic engineering protocol</div></td>
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<tr><td><br>The Mammalian team created lines of cells using lentiviruses for transfection.<br><br>
<div class="outline">
<div class="outline">
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  <a href="#htd">1 HTD Preparation Protocol</a><br>
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  <a href="#cpt">1 Calcium Phosphate Transfection</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#mixprep">1.1 PDMS Mixture Preparation</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#before">1.1 Preparation</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#pour">1.2 PDMS Pouring</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#prot">1.2 Protocol</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#baking">1.3 PDMS Baking</a><br>
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  <a href="#filter">2 Filter and Concentrate Virus</a><br>
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&nbsp;&nbsp;&nbsp;&nbsp;<a href="#punch">1.4 PDMS-Device Punching and Bonding</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#proto">2.1 Protocol</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#device">1.5 PDMS Device Bonding</a><br>
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  <a href="#materials">3 Materials Needed</a><br>
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&nbsp;&nbsp;&nbsp;&nbsp;<a href="#coat">1.6 PDL coating</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#hbss">3.1 HBSS Buffer</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#cf">1.7 Collagen filling</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#cacl">3.2 Calcium Chloride Solution</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#seed">1.8 Cell Seeding</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#freeze">3.3 Freezing medium</a><br>
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<a href="#deflection">2 Protocol for Deflection Experiments</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#dmem">3.4 Supplemented DMEM</a><br>
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&nbsp;&nbsp;&nbsp;&nbsp;<a href="#tubing">2.1 Tubing Setup Details</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#medium">2.2 Adding Medium to Channels</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#valve">2.3 Connecting device to pressure valve</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#micro">2.4 Microcontroller details</a><br>
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</div></td><tr><td><br>
</div></td><tr><td><br>
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<div class="bodybaby" id="htd">Mammalian Genetic Engineering Protocol</div><br>
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<div class="bodybaby" id="cpt">Calcium Phosphate Transfection</div><br>
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<b class="bolded" id="pour">Calcium Phosphate Transfection</b><br>
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<b class="bolded" id="before">Beforehand:</b><br>
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<ul id="procedure" style="list-style-type: circle;">
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<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>
  <ul><li>Particles must be of the right size for cells to take them up, controlled by pH: exactly 7.06</li>
  <ul><li>Particles must be of the right size for cells to take them up, controlled by pH: exactly 7.06</li>
Line 55: Line 85:
  <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>
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<b class="bolded" id="baking">PDMS Baking</b><br>
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<div class="bodybaby" id="filter">Filter and Concentrate Virus</div><br>
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<b class="bolded" id="proto">Protocol</b><br>
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<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&deg;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>
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<div class="bodybaby" id="materials">Materials</div><br>
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<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&deg;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>1. Bake the poured wafers for 4-8 hours at 80C (recommended: 24hours to cross-link most of PDMS monomers)</li>
+
  <li>450mL DMEM +Glucose +glutamine</li>
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  <li>2. Detach by cutting with razor (carefully and slowly peel it off, starting from the edges and going in the circumferential direction, in order to avoid tearing posts apart)</li> </ul>
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  <li>50mL FBS</li>
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<br><br>
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<li>5mL Pen/Strep</li>
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<li>0.5mL Fungin</li>
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<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:
  • Combine CaCL2 and dilute Na2PO4 to get CaPO4 with DNA in it
    • Particles must be of the right size for cells to take them up, controlled by pH: exactly 7.06
    • Use each aliquot 1-2 times then throw away because air contact makes the pH drift.
  • To make lentiviruses, need:
    • Gagpol for packaging (on PDR)
    • membrane prot on pVSV-G
    • Desired DNA
  • Cells should be at 50-70% confluence
  • Seed cells the day before: For 150 mm dish, seed about 7million cells
  • Can start with just confluent dish:
    • Split cells into 2 (to get about 50%)
    • 30 mins for cells to adhere (5-6h to spread out)
  • Coat dishes with gelatin for lentivirus production => cells detach less
  • Controls
    • No cells (see precipitate, like fine snow on cells)
    • Just carrier DNA (any plasmid)

Protocol
  • 1. Mix DNA (different plasmids in right amounts) with CaCl2
  • 2. Quickly vortex in 14 mL tube
  • 3. Add water to ~1.5 mL total volume
  • 4. Must saturate water with air - vortex without cap for 20-30 sec
  • 5. Make 1.5 mL HBSS aliquot
  • 6. For each tfxn, we now have 1 tube HBSS and 1 tube DNA+CaCl2+H2O in 14 mL tubes
  • 7. Add DNA solution dropwise to HBSS - 10-20 sec total - while vortexing solution. Use 2mL pipette for adding
  • 8. Immediately suck up into 5mL pipette and stop reaction by loading onto cells (spread by pipetting onto medium, no need to shake
  • 9. Check precipitation under microscope (small black dots between cells)
  • 10. Put cells back into incubator
  • 11. Next morning, change medium
  • 12. 1 and again 2 days after: Harvest 20mL (i.e. all the medium) to filter and centrifuge to obtain virus.

Filter and Concentrate Virus

Protocol
  • 1. Start with 20 + 20 mL culture supernatant
  • 2. Ultracentrifuge tubes (kept on shelf in tissue culture room) may contain residual bleach
    • Wash 2x with 70% EtOH
    • Wash 1x with medium
  • 3. Filter through 0.45 um (0.2 um can be used) Corning vacuum filter into 50 mL tube into ultracentrifuge tube
  • 4. Everything that contacts the virus including tips and plates must be bleached
  • 5. Spray filters with 10% bleach
  • 6. After filtered supernatant, add to each tube 4-5 mL sterile 20% w/v sucrose in PBS solution
    • Purpose of sucrose is to remove excess VSVG protein and dampen impact of particles
    • Phases will mix a little
  • 7. Balance weight with PBS to within <5mg
  • 8. Ultracentrifugation
    • Speed: 45,000 RCF (g)
    • Rotof: JA 25.50 (max 8 tubes)
    • Time: 3h
    • Accel: slow
    • Decel: OFF
    • Temp: 4°C
  • 9. When finished, cannot wait too long or pellet detaches
  • 10. Mark pellet, then aspirate supernatant
  • 11. Resuspend in 100uL PBS (or HBSS) for total of ~150 uL

Materials

HBSS Buffer (500 mL)
    • 8.0g NaCl
    • 0.37g KCl
    • 106.5mg Na2HPO4 (anhydrous, 201.1 mg if 7x H2O)
    • 1.0g dextrose
    • 5.0g Hepes
  • 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).
  • Filter through a 0.2 um filter, aliquot into 15 mL plastic tubes (12mL/tube). Can be kept at -20 for a few months.

Calcium Chloride Solution (50mL)
  • 5M CaCl2 (18.4g CaCl2*2H2O in 50mL)
  • Keep at +4°C

Freezing medium
  • 12mL FCS
  • 15mL DMEM (no antibiotics, directly as supplied)
  • 3mL DMSO
  • TOTAL: 30mL

Supplemented DMEM
  • 450mL DMEM +Glucose +glutamine
  • 50mL FBS
  • 5mL Pen/Strep
  • 0.5mL Fungin
  • TOTAL: 500mL