Difference between revisions of "Team:US AFRL CarrollHS/Experiments"

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<li>Thaw 50 µL vial of BL21 cells on ice</li>
 
<li>Thaw 50 µL vial of BL21 cells on ice</li>
 
<li>Inoculate 2µL DNA into 50µL BL21 cells</li>
 
<li>Inoculate 2µL DNA into 50µL BL21 cells</li>
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<ul>
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<ul class="real">
 
<li>incubate on ice for 30 minutes</li>
 
<li>incubate on ice for 30 minutes</li>
 
<li>heat shock at 42℃ for 10 seconds</li>
 
<li>heat shock at 42℃ for 10 seconds</li>
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</ul>
 
</ul>
 
<li>Inoculate 250µL LB into vial</li>
 
<li>Inoculate 250µL LB into vial</li>
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<ul>
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<ul class="real">
 
<li>incubate at 37℃ with shaking for 1.5 hours</li>
 
<li>incubate at 37℃ with shaking for 1.5 hours</li>
 
</ul>
 
</ul>
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<li>Inoculate 50 µL JM109 cells into Falcon tube</li>
 
<li>Inoculate 50 µL JM109 cells into Falcon tube</li>
 
<li>Inoculate 2µL DNA into 50µL JM109 cells</li>
 
<li>Inoculate 2µL DNA into 50µL JM109 cells</li>
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<ul>
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<ul class="real">
 
<li>incubate on ice for 20m</li>
 
<li>incubate on ice for 20m</li>
 
<li>heat shock at 42℃ for 45 seconds</li>
 
<li>heat shock at 42℃ for 45 seconds</li>
 
</ul>
 
</ul>
 
<li>Inoculate 950µL LB into vial</li>
 
<li>Inoculate 950µL LB into vial</li>
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<ul>
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<ul class="real">
 
<li>incubate at 37℃ with shaking for 1.5 hours</li>
 
<li>incubate at 37℃ with shaking for 1.5 hours</li>
 
</ul>
 
</ul>
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<li>Use ethanol-wiped scalpel to excise appropriate bands</li>
 
<li>Use ethanol-wiped scalpel to excise appropriate bands</li>
 
<li>Mix and incubate at 50°C for 10 min</li>
 
<li>Mix and incubate at 50°C for 10 min</li>
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<ul>
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<ul class="real">
 
<li>Vortex every 2 mins until gel slice is completely dissolved</li>
 
<li>Vortex every 2 mins until gel slice is completely dissolved</li>
 
</ul>
 
</ul>
 
<li>Place nucleospin column into 2mL collection tube</li>
 
<li>Place nucleospin column into 2mL collection tube</li>
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<ul>
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<ul class="real">
 
<li>Add sample</li>
 
<li>Add sample</li>
 
<li>Centrifuge at 11,000 xg for 1 minute</li>
 
<li>Centrifuge at 11,000 xg for 1 minute</li>
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  </ul>
 
  </ul>
 
<li>Add 700 mL Buffer NT3</li>
 
<li>Add 700 mL Buffer NT3</li>
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<ul>
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<ul class="real">
 
<li>Centrifuge at 11,000 xg for 1 minute</li>
 
<li>Centrifuge at 11,000 xg for 1 minute</li>
 
<li>Discard throughflow and place column back in same tube</li>
 
<li>Discard throughflow and place column back in same tube</li>
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<li>Repeat step 4</li>
 
<li>Repeat step 4</li>
 
<li>Centrifuge at 11,000 xg for another minute</li>
 
<li>Centrifuge at 11,000 xg for another minute</li>
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<ul>
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<ul class="real">
 
<li>To remove buffer</li>
 
<li>To remove buffer</li>
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<ul>
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<ul class="real">
 
<li>Discard throughflow</li>
 
<li>Discard throughflow</li>
 
<li>Centrifuge at 11,00 xg for add minute</li>
 
<li>Centrifuge at 11,00 xg for add minute</li>
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</ul>
 
</ul>
 
<li>Add 25 mL prewarmed (50°C) Buffer NE</li>
 
<li>Add 25 mL prewarmed (50°C) Buffer NE</li>
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<ul>
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<ul class="real">
 
<li>Incubate at 50°C for 5 min</li>
 
<li>Incubate at 50°C for 5 min</li>
 
<li>Centrifuge at 50 xg for 1 minute</li>
 
<li>Centrifuge at 50 xg for 1 minute</li>

Revision as of 23:44, 24 October 2017


Experiment

Transformations

BI21 Cells:

  1. Thaw 50 µL vial of BL21 cells on ice
  2. Inoculate 2µL DNA into 50µL BL21 cells
    • incubate on ice for 30 minutes
    • heat shock at 42℃ for 10 seconds
    • incubate on ice for 5m
  3. Inoculate 250µL LB into vial
    • incubate at 37℃ with shaking for 1.5 hours
  4. Plate 1x/9x

JM109 Cells:

  1. Thaw vial of JM109 cells on ice
  2. Label 14 mL Falcon tube & plane on ice
  3. Inoculate 50 µL JM109 cells into Falcon tube
  4. Inoculate 2µL DNA into 50µL JM109 cells
    • incubate on ice for 20m
    • heat shock at 42℃ for 45 seconds
  5. Inoculate 950µL LB into vial
    • incubate at 37℃ with shaking for 1.5 hours
  6. Plate 1x/9x

Electrophoresis/Gel Protocols

1% Agarose Electrophoresis Gel

  1. Add 0.35 g agarose to 35 mL 1x TAE in 100mL conical flask
  2. Heat in microwave for 35 seconds
  3. Swirl and heat in microwave for 25 seconds
  4. Swirl and heat in microwave for 15 seconds
  5. Swirl and heat in microwave for 7 seconds
  6. Swirl and ensure no ‘floaties’
  7. Cool outside of conical flask with water until ‘hand not’
  8. Pour into gel mold and add well comb

Gel Extraction

  1. Use ethanol-wiped scalpel to excise appropriate bands
  2. Mix and incubate at 50°C for 10 min
    • Vortex every 2 mins until gel slice is completely dissolved
  3. Place nucleospin column into 2mL collection tube
    • Add sample
    • Centrifuge at 11,000 xg for 1 minute
    • Discard throughflow and place column back in same tube
  4. Add 700 mL Buffer NT3
    • Centrifuge at 11,000 xg for 1 minute
    • Discard throughflow and place column back in same tube
  5. Repeat step 4
  6. Centrifuge at 11,000 xg for another minute
    • To remove buffer
      • Discard throughflow
      • Centrifuge at 11,00 xg for add minute
      • Place column in a clean, labelled 1.5 mL tube
  7. Add 25 mL prewarmed (50°C) Buffer NE
    • Incubate at 50°C for 5 min
    • Centrifuge at 50 xg for 1 minute
    • Centrifuge at 11,000 xg for 1 minute