Difference between revisions of "Team:Dartmouth/Protocols"
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<h1>Pf0-1 Pseudomonas Fluorescens Electrocompetency Prep</h1> | <h1>Pf0-1 Pseudomonas Fluorescens Electrocompetency Prep</h1> | ||
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<li>Repeat resuspension and centrifuge step</li> | <li>Repeat resuspension and centrifuge step</li> | ||
<li>Resuspend cell pellet with 50 uL or 100 uL 300 mM sucrose in water solution</li> | <li>Resuspend cell pellet with 50 uL or 100 uL 300 mM sucrose in water solution</li> | ||
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| + | <h2>Pf0-1 Pseudomonas Fluorescens Electroporation</h2> | ||
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| + | <li>Retrieve a 50 uL or 100 uL vial of electrocompentence prepped cells</li> | ||
| + | <li>Add 2 uL of DNA directly into vial of cells and mix by flicking</li> | ||
| + | <li>Transfer mixture to a 2 mm Electroporation cuvette</li> | ||
| + | <li>On BioRad Gene Pulser, pulse with 2.5 kV, 200 Ohm, 25 uF settings</li> | ||
</ul> | </ul> | ||
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Latest revision as of 04:00, 2 November 2017
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Pf0-1 Pseudomonas Fluorescens Electrocompetency Prep
- Innoculate 6 mL of liquid LB or Pseudomonas Minimal Media with Pf0-1 and grow overnight
- Split overnight into 1.5 mL aliquots and centrifuge at 16,000x g, discard supernatant
- Resuspend cell pellet with 1 mL of 300 mM sucrose in water solution
- Centrifuge at 16,000x g and discard supernatant
- Repeat resuspension and centrifuge step
- Resuspend cell pellet with 50 uL or 100 uL 300 mM sucrose in water solution
Pf0-1 Pseudomonas Fluorescens Electroporation
- Retrieve a 50 uL or 100 uL vial of electrocompentence prepped cells
- Add 2 uL of DNA directly into vial of cells and mix by flicking
- Transfer mixture to a 2 mm Electroporation cuvette
- On BioRad Gene Pulser, pulse with 2.5 kV, 200 Ohm, 25 uF settings
