Team:MIT mge

From 2010.igem.org

(Difference between revisions)
Line 57: Line 57:
</ul>
</ul>
<b class="bolded" id="pour">Calcium Phosphate Transfection</b><br>
<b class="bolded" id="pour">Calcium Phosphate Transfection</b><br>
-
<ul id="procedure" style="list-style-type: decimal;">
+
<ul id="procedure">
-
  <li>Mix DNA (different plasmids in right amounts) with CaCl2</li>
+
  <li>1. Mix DNA (different plasmids in right amounts) with CaCl2</li>
-
  <li>Quickly vortex in 14 mL tube</li>
+
  <li>2. Quickly vortex in 14 mL tube</li>
-
  <li>Add water to ~1.5 mL total volume</li>
+
  <li>3. Add water to ~1.5 mL total volume</li>
-
  <li>Must saturate water with air - vortex without cap for 20-30 sec</li>
+
  <li>4. Must saturate water with air - vortex without cap for 20-30 sec</li>
-
  <li>Make 1.5 mL HBSS aliquot</li>
+
  <li>5. Make 1.5 mL HBSS aliquot</li>
-
  <li>For each tfxn, we now have 1 tube HBSS and 1 tube DNA+CaCl2+H2O in 14 mL tubes</li>
+
  <li>6. For each tfxn, we now have 1 tube HBSS and 1 tube DNA+CaCl2+H2O in 14 mL tubes</li>
-
  <li>Add DNA solution dropwise to HBSS - 10-20 sec total - while vortexing solution. Use 2mL pipette for adding</li>
+
  <li>7. Add DNA solution dropwise to HBSS - 10-20 sec total - while vortexing solution. Use 2mL pipette for adding</li>
-
  <li>Immediately suck up into 5mL pipette and stop reaction by loading onto cells (spread by pipetting onto medium, no need to shake</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>Check precipitation under microscope (small black dots between cells)</li>
+
  <li>9. Check precipitation under microscope (small black dots between cells)</li>
-
  <li>Put cells back into incubator</li>
+
  <li>10. Put cells back into incubator</li>
-
  <li>Next morning, change medium</li>
+
  <li>11. Next morning, change medium</li>
-
  <li>1 and again 2 days after: Harvest 20mL (i.e. all the medium) to filter and centrifuge to obtain virus.</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>

Revision as of 17:24, 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 microfluidic protocol

The Mammalian team focused on creating a pressure-sensitive promoter and creating a standard protocol for mammalian genes.

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

Mammalian Genetic Engineering Protocol

Before Calcium Phosphate Transfection:
  • 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)
Calcium Phosphate Transfection
  • 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.

PDMS Baking
  • 1. Bake the poured wafers for 4-8 hours at 80C (recommended: 24hours to cross-link most of PDMS monomers)
  • 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)

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