Team:MIT mge
From 2010.igem.org
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- | <b class="bolded" id="baking"> | + | <b class="bolded" id="baking">Filter and Concentrate Virus</b><br> |
<ul id="procedure"> | <ul id="procedure"> | ||
- | <li>1. | + | <li>1. Start with 20 + 20 mL culture supernatant</li> |
- | <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°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><br> | <br><br> | ||
Revision as of 17:34, 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:
Filter and Concentrate Virus
|