Team:Stockholm/Protocols

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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.

Competent cells (Andreas & Mimmi)

Based and modified from the [http://openwetware.org/wiki/TOP10_chemically_competent_cells Top10 protocol by the Knight lab]

Materials

CCMB80 buffer

• 10 mM KOAc pH 7.0
• 80 mM CaCl2.2H2O
• 20 mM MnCl2
• 10 mM MgCl2.6H2O
• 10% glycerol

Procedures

1. Set an ON starter culture by inoculating 5 ml LB and incubating ON in 37°C with 250 rpm rotary shaking.
2. Inoculate 250 ml new LB with 2 ml from the ON culture and grow in 30°C, 250 rpm until an OD(600) of ≈0.3.
   • Use a large, 1 l E-flask.
3. Spin down cells at 3000 x g for 10 min in 4°C.
4. Remove supernatant and resuspend cells in 80 ml ice-cold CCMB80 buffer. Keep cells on ice for 20 min.
5. Spin down cells again with same settings. Resuspend in 10 ml new CCMB80 buffer and keep on ice for 20 min.
6. Aliquot 100 ul samples of competent cells into 1.5 ml vials and store in -80°C, or transform immediately.

1. 250 ml LB inoculated with 2 ml from ON culture
   • Grown at 28°C until an OD(600) of ≈0.3
2. Cells spun down at 3000x g for 10 min in 4°C
3. Cells resuspended in 80 ml ice-cold CCMB80 buffer and kept on ice for 20 min:
4. Cells spun down with same settings
5. Cells resuspended in 10 ml new CCMB80 and kept on ice for 20 min
6. Competent cells aliquoted in 100ul aliquots and stored in -80°C.

Transformation (Andreas)

1. Add 1 ul plasmid to your 100 ul thawed, competent cells of choice. Hold cells on ice for 30 min.
2. Heat-shock cells for 55 sec in 42°C. Return to ice.
3. 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).
4. Spin down cells at full speed (≈13.000 x g) for 15 sec.
5. Remove 900 ul from the supernatant and gently resuspend pellet in remaining 100 ul.
6. Plate 100 ul cells onto an LB agar plate with appropriate antibiotic(s).