Difference between revisions of "Team:UMBC-Maryland/Experiments"

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<h5>Agarose Gel</h5>
 
<h5>Agarose Gel</h5>
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Electrophoresis buffer contents:
 
Electrophoresis buffer contents:
 
<li>10 mL 50x TAE</li>
 
<li>10 mL 50x TAE</li>

Revision as of 18:42, 14 August 2015

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Experiments & Protocols

Agarose Gel
Electrophoresis buffer contents:
  • 10 mL 50x TAE
  • 490 mL deionized water

    • Contents of Agarose gel:
  • 1g Agarose (1%) or 2g Agarose (2%)
  • 100 mL Electrophoresis buffer

    1. Mix agarose and electrophoresis buffer and microwave for 1.5 minutes.
    2. When the solution is sufficiently cool, add 6 µL of EtBr.
    3. Pour the solution into a bed with a comb levely placed within. Clear the bubbles from the surface and wait for the gel to polymerize.Mix loading dye and sample in a 1:5 ratio and insert samples into wells. Place 10 µL of DNA ladder into the first well.

    Polymerase Chain Reaction
    We used Quick-Load® Taq 2X Master Mix, New England Biolabs according to the manufacturer's instructions for PCR.

    DNA Assembly and Cloning
    We used Gibson Assembly® Protocol (E5510), New England Biolabsaccording to the manufacturer's instructions for cloning.

    Plasmid Extraction
    We used NucleoSpin® Plasmid Miniprep kit, Macherey-Nagel according to the manufacturer's instructions for our minipreps.

    Restriction Digest
  • 8 μL 2.1 Buffer
  • 5 μL DNA
  • 2 μL EcoR1
  • 2 μL Pst1
    • Incubate at 37°C for 1 hour
    Preparing Copper Stock Solution (1M)
    1. Transfer 50mL of water into a volumetric flask
    2. Add 6.24g of Copper Sulfate Pentahydrate into flask
    3. Dilute Copper solution with water in a 1:4 ratio

    Inoculation Experiments
    1. In sterile conditions, prepare 8 flasks with 25 mL LB solution, labeled Control 0 µM Cu, Control 0.5 µM Cu, Control 1 µM Cu, Control 4 µM Cu, Retriever 0 µM Cu, Retriever 0.5 µM Cu, Retriever 1 µM Cu, and Retriever 4 µM Cu.
    2. Add appropriate amounts of copper sulfate solution to each flask.
    3. Add 9.375 µL chloramphenicol from 34 mg/ml stock to Retriever flasks.
    4. Add control cells to control flasks and transformed cells to Retriever flasks
    5. Place all flasks in the shaker at 37°C and 250 rpm.
    6. Each hour, beginning at the time of inoculation, take a sample from each flask.
    7. Take measurements for optical density, copper concentration, and protein translation level.
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