Difference between revisions of "Team:Washington/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

• 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.

• Cut strips of Whatman 114 filter paper to dimensions 3cm x 4cm
• Cut 2cm x 2cm pieces of PDMS sheet/li>
• 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./li>
• Autoclave devices in a sterile container on a dry cycle/li>