Team:MIT mge
From 2010.igem.org
(Difference between revisions)
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</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">Mammalian Genetic Engineering Protocol</div><br> | ||
- | <b class="bolded" id="pour">Calcium Phosphate Transfection</b><br> | + | <b class="bolded" id="pour">Before Calcium Phosphate Transfection:</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> | ||
+ | <b class="bolded" id="pour">Calcium Phosphate Transfection</b><br> | ||
+ | <ul id="procedure" style="list-style-type: decimal;"> | ||
+ | <li>Mix DNA (different plasmids in right amounts) with CaCl2</li> | ||
+ | <li>Quickly vortex in 14 mL tube</li> | ||
+ | <li>Add water to ~1.5 mL total volume</li> | ||
+ | <li>Must saturate water with air - vortex without cap for 20-30 sec</li> | ||
+ | <li>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>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>Check precipitation under microscope (small black dots between cells)</li> | ||
+ | <li>Put cells back into incubator</li> | ||
+ | <li>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> | ||
</ul> | </ul> | ||
<br> | <br> |
Revision as of 17:23, 16 October 2010
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:
PDMS Baking
|