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