Team:MIT mge
From 2010.igem.org
(Difference between revisions)
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<tr><td><br>The Mammalian team created lines of cells using lentiviruses for transfection.<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="# | + | <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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<li></li> | <li></li> | ||
</ul><br> | </ul><br> | ||
- | <b class="bolded" id=" | + | <b class="bolded" id="prot">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> | ||
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</ul> | </ul> | ||
<br> | <br> | ||
- | <div class="bodybaby" id=" | + | <div class="bodybaby" id="filter">Filter and Concentrate Virus</div><br> |
- | <b class="bolded" id=" | + | <b class="bolded" id="proto">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> | ||
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<li>11. Resuspend in 100uL PBS (or HBSS) for total of ~150 uL</li> | <li>11. Resuspend in 100uL PBS (or HBSS) for total of ~150 uL</li> | ||
</ul> | </ul> | ||
- | <br><br> | + | <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></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> | ||
</td> | </td> |
Revision as of 17:44, 16 October 2010
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. |
Calcium Phosphate Transfection Beforehand:
Protocol
Filter and Concentrate Virus Protocol
Materials HBSS Buffer (500 mL)
|