Difference between revisions of "Team:Washington/Protocols"

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<p>Put yeast plate to a blue light imager.</p>
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<p>Note differences in brightness between yeast colonies</p>
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<li>Put yeast plate to a blue light imager.</li>
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<li>Note differences in brightness between yeast colonies</li>
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<h2>Assay:</h2>
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<p>Assay:</p>
 
<p>Fill 96 well plate with 250 uL of cell cultures<p>
 
<p>Fill 96 well plate with 250 uL of cell cultures<p>
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<h2>Flow Cytometer:</h2>
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<p>Flow Cytometer:</p>
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<p>Set a bottom cutoff of 10,000 units
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<p>Excitation: 515 nm</p>
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<li>Set a bottom cutoff of 10,000 units</li>
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<p>Emission: 530 nm</p>
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<li>Excitation: 515 nm</li>
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<li>Emission: 530 nm</li>
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Revision as of 02:03, 18 September 2015



Protocols

Digest E. Coli plasmid using PmeI restriction enzyme

  • 1 ug of DNA
  • 5 uL of 10x NEB CutSmart buffer
  • 1 uL of restriction enzyme
  • Fill to 50 uL with water
  • Incubate at 37 C for 15 minutes (1 hour if not using TimeSaver buffer)
  • Heat inactivate at 65 C for 20 minutes
  • Agarose gel purify (optional)

Salmon Sperm Transformation

  • Grow a yeast overnight
  • Check OD of culture. 0.5-0.6 are the preferred readings, if the reading is lower, wait for longer growth, if the reading is higher, dilute the sample.
  • Spin down 10 ml of cells per transformation.
  • Decant supernatant and wash with 10 ml ddH2O. Vortex to resuspend and spin down.
  • Remove the supernatant.
  • Resuspend cells in 300 uL .1 M LiOAc. Transfer to a 1.5 mL tube.
  • Incubate at 30 C for 15 min
  • Put salmon sperm DNA in boiling water for 5 minutes. Cool immediately on ice.
  • Spin down cells and remove supernatant.
  • Add the following in order:
    1. 240 uL 50% PEG
    2. 36 uL 1.0 M LioAc
    3. 10 uL salmon sperm DNA
    4. 34 uL DNA
    5. 40 uL ddH2O
    6. Final volume: 360 uL

50 mM Theophylline dissolved by DMSO

Replace occasionally due to possible interactions between theophylline & DMSO

  • Put yeast plate to a blue light imager.
  • Note differences in brightness between yeast colonies

Assay:

Fill 96 well plate with 250 uL of cell cultures

Flow Cytometer:

  • Set a bottom cutoff of 10,000 units
  • Excitation: 515 nm
  • Emission: 530 nm

Insert text

For commercial shellfish farmers and recreational hunters alike, marine biotoxins pose a significant threat to health and welfare. With this project, we aim to create an inexpensive and easy-to-use test kit for the detection of the shellfish toxin okadaic acid using engineered yeast strains and DNA aptamers on a paper device. We also hope that this project paves the way for a new class of biosensors capable of detecting a wide range of small molecules.