Team:UMBC-Maryland/Experiments

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.

    SDS-PAGE Gel
    We used Mini-PROTEAN® TGX™ Precast Polyacrylamide Gels, BIO-RAD according to the manufacturer's instructions for SDS-Page gels.

    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 NEBuilder® HiFi DNA Assembly, 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
  • 1.5 μL of 10x 3.1 Buffer
  • 3 μL DNA
  • 10.1 μL D-Water
  • 0.2 μL EcoR1
  • 0.2 μL Pst1
    • Incubate at 37°C for 30 minutes

    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
    In sterile conditions, prepare 8 flasks with 25 mL LB solution with control and transformed cells. Designate 4 flasks for each strain. Designate 1 flask for each strain for 4 different initial copper sulfate concentrations. Label each flask with appropriate strain and concentration. Ex. Retriever1 4 mM, Control 2 mM.
    1. Add appropriate amounts of copper sulfate solution to each flask.
    2. Add 9.375 µL chloramphenicol from 34 mg/ml stock to Retriever (transformed) flasks.
    3. Add control cells to control flasks and transformed cells to Retriever flasks
    4. Place all flasks in the shaker at 37°C and 250 rpm.
    5. Each hour, beginning at the time of inoculation, take a sample from each flask.
    6. Take measurements for optical density, copper concentration, and protein translation level.
    Taking hourly timepoints
    1. Prepare two microcentrifuge tubes for each flask per timepoint. Label each pair of tubes with the hour timepoint, strain, and initial copper concentration. On one of the tubes for each flask for this timepoint, mark "Cu" on the side to represent copper measurement. An example label for a sample with Retriever 1 and initial copper concentration 0 mM at hour 3 would be "Ret1, 0 mM, h 3" with comma denoting separate lines.
    2. Turn off shaking incubator and remove flasks.
    3. Using sterile technique so as to keep unwanted cells from entering the flasks, pipette 1 ml from each flask into the respective microcentrifuge tube that does not have "Cu" on the side.
    4. Place flasks inside shaking incubator and set rotations to 250 rpm.
    5. Centrifuge microcentrifuge tubes for 5 minutes at 14,000 g.
    6. Pipette supernatant from centrifuged tubes into respective tubes marked with "Cu" for copper measurement.
    7. Store filled copper measurement tubes inside refrigerator.
    8. Resuspend pelleted cells in 1 ml of LB and vortex.
    9. If recording first timepoint, blank biospectrophotometer with 1 ml sample of pure, sterile LB.
    10. Measure the cell density of each resuspended microcentrifuge tube at 600 nm and record absorbance.
    11. Centrifuge microcentrifuge tubes for 5 minutes at 14,000 g.
    12. Remove and discard supernatant.
    13. Resuspend pelleted cells in 100 ul 1:1 Laemmli buffer and vortex.
    14. Store microcentrifuge tubes in freezer for an SDS gel

    Measuring Copper Concentration with Atomic Absorption Spectroscopy
    1. Turn on fuel and light flame inside AAS machine.
    2. Set measurement to the wavelength of copper
    3. Prepare 1 ml samples for 10 copper concentrations within range being tested. Measure and record absorbance at these concentrations to prepare a calibration curve.
    4. Pipette 1 ml timepoint samples into clean glass tube.
    5. Insert collection tube into sample.
    6. Press the "read sample" button several times and wait 30 seconds before recording absorbance value.

    Measuring Copper Concentration with 96 Well Plate Reader
    1. Label on paper which samples will go into which wells.
    2. Prepare the following calibration curve samples with copper sulfate in LB: 0, .1, .3, .5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 mM.
    3. Pipette 100 ul of each sample into 96 well plate.
    4. Create a new protocol and experiment to measure the absorbance in all wells at 620 nm.
    5. Create a calibration curve for concentration as a function of absorbance using the absorbance readings of the known samples.
    6. Calculate the concentrations of the measured samples using a linear fit of this calibration curve. If the calibration curve is not linear, a different wavelength should be used.