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Team:Stockholm/Protocols
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11. Snapfreeze 100 mikroL aliquots in ice-cold Epps (in pre-chilled blocks). Store in -80 degree C freezer. | 11. Snapfreeze 100 mikroL aliquots in ice-cold Epps (in pre-chilled blocks). Store in -80 degree C freezer. | ||
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| + | ==Transformation (Andreas)== | ||
| + | # Add 1 ul plasmid to your 100 ul thawed, competent cells of choice. Hold cells on ice for 30 min. | ||
| + | # Heat-shock cells for 55 sec in 42°C. Return to ice. | ||
| + | # Add 900 ul LB medium and grow cells in 37°C, 250 rpm for 1 hour. | ||
| + | #* This allows the cell to start expressing antibiotic resistance gene(s). | ||
| + | # Spin down cells at full speed (≈13.000 x ''g'') for 15 sec. | ||
| + | # Remove 900 ul from the supernatant and gently resuspend pellet in remaining 100 ul. | ||
| + | # Plate 100 ul cells onto an LB agar plate with appropriate antibiotic(s). | ||
Revision as of 08:40, 6 July 2010
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Competent cells (From Morten Nørholm at the Department of Biochemistry & Biophysics Stockholm University)
1. Add 5 ml LB in two 50 ml falcon tubes. Add the top of a tip bacteria into the two 5 ml LB. Grow ON in shake incubator 37 degree C.
2. Subculture each 5 ml of starterculture into two 400 ml pre-warmed LB. Grow at 37 degree C until OD reaches 0.6.
3. Put cells on ice for 20 min.
4. Harvest cells at 4000 rpm for 20 min, 4 degree C.
5. Discard supernatant if it looks clear (or spinn longer if it is cloudy).
6. Resuspend pellet carefully in 500 50 mM CaCl2 for a 1000 ml cell culture (1/2 the orifinal volume).
7. Put cells on ice 20 min.
8. Repeat step 5.
9. Resuspend cells in 16 ml CaCl2 + 15% Glycerol for a 800 ml starter culture (1:50 volume)
10. Put metal blocks in -80 degree C.
11. Snapfreeze 100 mikroL aliquots in ice-cold Epps (in pre-chilled blocks). Store in -80 degree C freezer.
Transformation (Andreas)
- Add 1 ul plasmid to your 100 ul thawed, competent cells of choice. Hold cells on ice for 30 min.
- Heat-shock cells for 55 sec in 42°C. Return to ice.
- Add 900 ul LB medium and grow cells in 37°C, 250 rpm for 1 hour.
- This allows the cell to start expressing antibiotic resistance gene(s).
- Spin down cells at full speed (≈13.000 x g) for 15 sec.
- Remove 900 ul from the supernatant and gently resuspend pellet in remaining 100 ul.
- Plate 100 ul cells onto an LB agar plate with appropriate antibiotic(s).
