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

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<h2>Methodology</h2>
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<h2>Electrophoresis/Gel Protocols</h2>
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<h3>1% Agarose Electrophoresis Gel</h3>
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<ol>
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<li>Add 0.35 g agarose to 35 mL 1x TAE in 100mL conical flask</li>
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<li>Heat in microwave for 35 seconds</li>
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<li>Swirl and heat in microwave for 25 seconds</li>
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<li>Swirl and heat in microwave for 15 seconds</li>
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<li>Swirl and heat in microwave for 7 seconds</li>
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<li>Swirl and ensure no ‘floaties’ </li>
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<li>Cool outside of conical flask with water until ‘hand not’</li>
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<li>Pour into gel mold and add well comb</li>
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</ol>
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<h3></h3>
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Revision as of 23:27, 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