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

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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

1.5 μL of 10x 3.1 Buffer

3 μL DNA

10.1 μL D-Water

.2 μL EcoR1

.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

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.