Difference between revisions of "Team:UMBC-Maryland/Experiments"
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<li>100 mL Electrophoresis buffer</li> | <li>100 mL Electrophoresis buffer</li> | ||
<br> | <br> | ||
− | + | 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. | |
− | + | 4.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. | |
<br> | <br> | ||
− | <h5> | + | <h5>Polymerase Chain Reaction</h5> |
− | We used <a href=" | + | We used <a href="https://www.neb.com/products/m0271-quick-load-taq-2x-master-mix">Quick-Load® Taq 2X Master Mix, New England Biolabs</a> according to the manufacturer's instructions for PCR. |
<br></br> | <br></br> | ||
<h5>DNA Assembly and Cloning</h5> | <h5>DNA Assembly and Cloning</h5> | ||
We used <a href="https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510">Gibson Assembly® Protocol (E5510), New England Biolabs</a>according to the manufacturer's instructions for cloning. | We used <a href="https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510">Gibson Assembly® Protocol (E5510), New England Biolabs</a>according to the manufacturer's instructions for cloning. | ||
+ | <br></br> | ||
+ | <h5>Plasmid Extraction</h5> | ||
+ | We used <a href="http://www.mn-net.com/ProductsBioanalysis/DNAandRNApurification/PlasmidDNApurificationeasyfastreliable/NucleoSpinPlasmidplasmidMiniprepkit/tabid/1379/language/en-US/Default.aspx">NucleoSpin® Plasmid Miniprep kit, Macherey-Nagel </a> according to the manufacturer's instructions for our minipreps. | ||
<br></br> | <br></br> | ||
<h5>Restriction Digest</h5> | <h5>Restriction Digest</h5> | ||
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<li>2 μL EcoR1</li> | <li>2 μL EcoR1</li> | ||
<li>2 μL Pst1</li> | <li>2 μL Pst1</li> | ||
− | + | Incubate at 37°C for 1 hour | |
− | < | + | <br> |
+ | <h5>Preparing Copper Stock Solution (1M)</h5> | ||
+ | 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 | ||
+ | <br> | ||
<h5>Inoculation Experiments</h5> | <h5>Inoculation Experiments</h5> | ||
− | 1. In sterile conditions, prepare 8 flasks with 25 mL LB solution, | + | 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. | ||
<h5>What should this page contain?</h5> | <h5>What should this page contain?</h5> |
Revision as of 18:36, 14 August 2015
Experiments & Protocols
Agarose Gel
Electrophoresis buffer contents:Contents of Agarose gel:
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. 4.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)
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 ratioInoculation 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.What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project