Team:MIT mge

From 2010.igem.org

(Difference between revisions)
m
Line 20: Line 20:
<div id="unique" style="padding:5px; font-size: 14px; border: 1px solid black; margin:5px;">
<div id="unique" style="padding:5px; font-size: 14px; border: 1px solid black; margin:5px;">
-
<table width=70%><tr><td><div class="bodybaby">mammalian microfluidic protocol</div></td>
+
<table width=70%><tr><td><div class="bodybaby">mammalian genetic engineering protocol</div></td>
-
<tr><td><br>The Mammalian team focused on creating a pressure-sensitive promoter and creating a standard protocol for mammalian genes.<br><br>
+
<tr><td><br>The Mammalian team created lines of cells using lentiviruses for transfection.<br><br>
<div class="outline">
<div class="outline">
  <a href="#htd">1 HTD Preparation Protocol</a><br>
  <a href="#htd">1 HTD Preparation Protocol</a><br>
Line 39: Line 39:
</div></td><tr><td><br>
</div></td><tr><td><br>
-
<div class="bodybaby" id="htd">Mammalian Genetic Engineering Protocol</div><br>
+
<div class="bodybaby" id="htd">Calcium Phosphate Transfection</div><br>
-
<b class="bolded" id="pour">Before Calcium Phosphate Transfection:</b><br>
+
<b class="bolded" id="pour">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>
Line 55: Line 55:
  <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>
+
</ul><br>
-
<b class="bolded" id="pour">Calcium Phosphate Transfection</b><br>
+
<b class="bolded" id="pour">Protocol</b><br>
<ul id="procedure">
<ul id="procedure">
  <li>1. Mix DNA (different plasmids in right amounts) with CaCl2</li>
  <li>1. Mix DNA (different plasmids in right amounts) with CaCl2</li>
Line 72: Line 72:
</ul>
</ul>
<br>
<br>
-
<b class="bolded" id="baking">Filter and Concentrate Virus</b><br>
+
<div class="bodybaby" id="htd">Filter and Concentrate Virus</div><br>
 +
<b class="bolded" id="baking">Protocol</b><br>
<ul id="procedure">
<ul id="procedure">
  <li>1. Start with 20 + 20 mL culture supernatant</li>
  <li>1. Start with 20 + 20 mL culture supernatant</li>

Revision as of 17:36, 16 October 2010

Methods

Bacterial Construction Protocol
Bacterial Experimental Protocol
Phage western blot
Mammalian Protocol
Microfluidic stress

Mammalian Protocol

Microfluidics Protocol
Genetic Engineering Protocol
mammalian genetic engineering protocol

The Mammalian team created lines of cells using lentiviruses for transfection.

1 HTD Preparation Protocol
    1.1 PDMS Mixture Preparation
    1.2 PDMS Pouring
    1.3 PDMS Baking
    1.4 PDMS-Device Punching and Bonding
    1.5 PDMS Device Bonding
    1.6 PDL coating
    1.7 Collagen filling
    1.8 Cell Seeding
2 Protocol for Deflection Experiments
    2.1 Tubing Setup Details
    2.2 Adding Medium to Channels
    2.3 Connecting device to pressure valve
    2.4 Microcontroller details

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

Retrieved from "http://2010.igem.org/Team:MIT_mge"