Team:UMBC-Maryland/Experiments
Experiments & Protocols
Agarose Gel
Electrophoresis buffer contents:Contents of Agarose gel:
- Mix agarose and electrophoresis buffer and microwave for 1.5 minutes.
- When the solution is sufficiently cool, add 6 µL of EtBr.
- 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
Preparing Copper Stock Solution (1M)
- Transfer 50mL of water into a volumetric flask
- Add 6.24g of Copper Sulfate Pentahydrate into flask
- Dilute Copper solution with water in a 1:4 ratio
Inoculation Experiments
- 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.
- Add appropriate amounts of copper sulfate solution to each flask.
- Add 9.375 µL chloramphenicol from 34 mg/ml stock to Retriever flasks.
- Add control cells to control flasks and transformed cells to Retriever flasks
- Place all flasks in the shaker at 37°C and 250 rpm.
- Each hour, beginning at the time of inoculation, take a sample from each flask.
- Take measurements for optical density, copper concentration, and protein translation level.