Difference between revisions of "Team:Washington/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:
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

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

Making 1mm-Thick Sheets of PDMS (Polydimethyl Siloxane)

• 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.

Making the Paper Device

• Cut strips of Whatman 114 filter paper to dimensions 3cm x 4cm
• Cut 2cm x 2cm pieces of PDMS sheet
• Use heat-resistant tape (such as electrical tape or 3M heat-resistant packing tape) to tape polymer squares to either side of the paper strip. On one side, tape around all four sides of the PDMS. On the other, leave the top open in order to insert media and cells.
• Autoclave devices in a sterile container on a dry cycle

Inserting cells and media

• Make either liquid or gel media with conditions specific for the intended yeast strain (note that liquid media may dry out fast)
• For liquid media, pipette no more than 100uL through the open side of the device, between the paper and the PDMS window
• For gel media, pipette no more than 300uL between the paper and PDMS
• Using a pipette tip or toothpick, insert a clump of cells and deposit them in the middle of the PDMS window. For gel media, it may be necessary to use a toothpick to poke a pathway through the gel in order to ensure cells can be deposited easily.
• Tape over the remaining side of the device to seal the cells and media inside

Running detection assays

• The molecule of interest can either be pipetted directly between the paper and PDMS along with the yeast media, or the device can be stood up in a solution of the molecule, which can travel to the yeast via wicking.

Yeast lysis on paper to view beta-Gal production

• Place yeast on liquid media inside a device.
• Pipette 30uL of 0.2% SDS and 10uL of 50mM X-gal directly to the cells, or place end of device in solution of 300uL of 0.2% SDS and 100uL X-gal
• When SDS is in contact with cells, massage PDMS window briefly to mix
• Allow devices to sit at 37C for 30 minutes to 1 hour

Observe blue color

• Theophylline detection on paper (assuming inducible Gal promoter)
• Load device with theophylline-detecting strain and c-ura media, with galactose as the sugar*
• Seal device
• Allow device to sit in a beaker of 3mL of 50mM theophylline solution for 6-10 hours.
• Observe fluorescence

*Alternatively, use media with no sugar and add galactose the same way as theophylline

Possible controls

• Strain that doesn’t produce theo aptamer with theophylline (negative control)
• Theo detection strain without theophylline (negative control)
• Strain that constitutively produces YFP with theophylline (positive control)
• Theo detection strain on arafinose or glucose c-ura media
• Theo detection strain on galactose c-ura with caffeine (no theophylline)

Auxin detection on paper

Possible controls

• Strain without auxin detection pathway with auxin (negative control)
• Strain with auxin detection pathway without auxin (negative control)
• Strain without gRNA, which constitutively produces beta-Gal/blue chromoprotein with auxin (positive control)