Team:Grinnell/Notebook/Protocols
From 2011.igem.org
Competent Cells
- Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5 x 10^8 cells/mL, OD600 = 0.3-0.5).
- Chill cells on ice for 15-120min.
- Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4° C. Discard supernatant.
- Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.
- Harvest cells by cetrifugation. Discard supernatant.
- Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80° C.
Plasmid Transformation
- Thaw 100μL aliquots of competent cells on ice.
- Add 10μL DNA to cells.
- Incubate tubes on ice for 30min.
- Incubate tubes at 42° C for 90sec.
- Incubate tubes on ice for 2min.
- Add 300μL LB to cells and incubate shaking at 37° C for 1hr.
- Spread cells on selective media
- Incubate plates overnight at 37° C.
Isolation of DNA for Colony PCR
GeneReleaser is a proprietary reagent that releases DNA from cells while sequestering cell lysis products that might inhibit DNA polymerases.- Resuspend the GeneReleaser through inversion, not vortexing. Add 20μL GeneReleaser to each PCR tube.
- Add cells from plates with a sterile pipette tip with 10μL of appropriate liquid media OR 10μL from overnight liquid culture.
- Run PCR tubes on following thermal cycle program:
- DNA will be in the clear liquid above the white precipitate at bottom of tube.
Temperature (°C) | Time (s) |
---|---|
65 | 30 |
8 | 30 |
65 | 90 |
97 | 180 |
8 | 60 |
65 | 180 |
97 | 60 |
65 | 60 |
80 | hold |
Agarose Gel Electrophoresis
- To make a 0.7% agarose content gel first add 0.21g agarose and then 30mL 1 X TBE buffer to a 250mL Erlenmeyer flask.
- Microwave until the solution boils, about 45-60sec. Let boil for 5sec. Then check for agarose that has not gone into solution and until until solution is homogeneous.
- Let solution sit until it is cool enough to touch and then add 2μL ethidium bromide using caution and swirl mixture.
- Set up gel tray and combs and pour gel until it is solidified, about 30min.
- Place gel in chamber oriented with positive electrode at the bottom of the gel and cover with 1X TBE.
- Add 5uL water, 5μL DNA, and 2μL 6X loading dye
- Remove the comb and load each sample along with 10μL of a 1kb ladder. Run at
- When loading dye has run to the end of the gel, remove gel.