Difference between revisions of "Team:Washington/Protocols"
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| + | <h2>Yeast Chromosome Integration</h2> | ||
<p>Digest E. Coli plasmid using PmeI restriction enzyme</p> | <p>Digest E. Coli plasmid using PmeI restriction enzyme</p> | ||
<ul list-style-image:url("https://static.igem.org/mediawiki/2015/e/e9/Replace_pixel_washington.png");> | <ul list-style-image:url("https://static.igem.org/mediawiki/2015/e/e9/Replace_pixel_washington.png");> | ||
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</ul> | </ul> | ||
| + | <h2>Theophylline Stock</h2> | ||
<p>50 mM Theophylline dissolved by DMSO</p> | <p>50 mM Theophylline dissolved by DMSO</p> | ||
<p>Replace occasionally due to possible interactions between theophylline & DMSO</p> | <p>Replace occasionally due to possible interactions between theophylline & DMSO</p> | ||
| + | <h2>Fluorescence Reading</h2> | ||
<ul list-style-type:none> | <ul list-style-type:none> | ||
<li>Put yeast plate to a blue light imager.</li> | <li>Put yeast plate to a blue light imager.</li> | ||
Revision as of 02:59, 18 September 2015
Protocols
Aptamer Protocols
Yeast Chromosome Integration
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:
- 240 uL 50% PEG
- 36 uL 1.0 M LioAc
- 10 uL salmon sperm DNA
- 34 uL DNA
- 40 uL ddH2O
- Final volume: 360 uL
Theophylline Stock
50 mM Theophylline dissolved by DMSO
Replace occasionally due to possible interactions between theophylline & DMSO
Fluorescence Reading
- 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.