Team:Washington/Protocols

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Protocols

Aptamer Protocols

• Yeast Chromosome Integration
• Theophylline Stock
• Fluorescence Reading

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

Paper Protocols

• 1mm Sheets of PDMS
• Making the Paper Device
• Inserting cells and media
• Running detection assays
• Viewing beta-Gal production
• Theophylline detection on paper
• Auxin detection on paper

• Obtain Sylgard 184 Silicone Elastomer Kit from Dow Corning
• Obtain cell culture plates to use as templates
• Calculate surface area of plate in order to determine the mass needed of PDMS
• Make a 1:10 mixture of activator to PDMS
• Stir well using a wood or plastic stick
• Pour polymer onto the top of the lid or bottom of the culture plate. Pour into the middle and work out towards the edges.
• Tilt plate to let PDMS flow to the edges of the plate.
• Remove bubbles, either by putting the sample in a vacuum or by popping the bubbles with the stirring stick
• Let polymer cure for 2 days in a flat place to ensure even distribution.

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.