Team:Stockholm/Protocols
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Contents |
Competent cells (Nina & Johan)
From Morten Nørholm at the Department of Biochemistry & Biophysics Stockholm University
- 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.
- Subculture each 5 ml of starterculture into two 400 ml pre-warmed LB. Grow at 37 degree C until OD reaches 0.6.
- Put cells on ice for 20 min.
- Harvest cells at 4000 rpm for 20 min, 4 degree C.
- Discard supernatant if it looks clear (or spinn longer if it is cloudy).
- Resuspend pellet carefully in 500 50 mM CaCl2 for a 1000 ml cell culture (1/2 the orifinal volume).
- Put cells on ice 20 min.
- Repeat step 5.
- Resuspend cells in 16 ml CaCl2 + 15% Glycerol for a 800 ml starter culture (1:50 volume)
- Put metal blocks in -80 degree C.
- 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 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
- Set an ON starter culture by inoculating 5 ml LB and incubating ON in 37°C with 250 rpm rotary shaking.
- 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.
- Spin down cells at 3000 x g for 10 min in 4°C.
- Remove supernatant and resuspend cells in 80 ml ice-cold CCMB80 buffer. Keep cells on ice for 20 min.
- Spin down cells again with same settings. Resuspend in 10 ml new CCMB80 buffer and keep on ice for 20 min.
- Aliquot 100 ul samples of competent cells into 1.5 ml vials and store in -80°C, or transform immediately.
Transformation (Nina & Johan)
From NEB 5-alpha competent E.coli (High Efficiency) NEW ENGLAND BioLabs
- Thaw a tube of NEB 5-alpha competent E.coli cells on ice for 10 min.
- Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix the cells and DNA.
- Place the mixture on ice for 30 min.
- Heat shock at 42°C for 30 sec.
- Place on ice for 5 min.
- Pipette 950 μl of room temp. SOC into the mixture.
- Place at 37°C for 30-60 min, 250 rpm.
- Warm selection plates to 37°C.
- Perform 10-fold serial dilutions in SOC (if necessary)
- Spread 50-100 μl of each sample onto a selection plate and incubate overnight at 37°C.
Transformation (Andreas & Mimmi)
- Add 1 ul plasmid to 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 cells 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).
Colony PCR verification (Andreas & Mimmi)
- Pick four colonies and resuspend each in 10 ul LB.
- Let incubate in RT while preparing PCR tubes.
- Prepare each illustra PuReTaq Ready-To-Go PCR (GE Healthcare) tube as follows:
- 1.5 ul 10uM forward primer
- 1.5 ul 10uM reverse primer
- 21 ul dH2O
- Adjust dH2O volume according to primer concentrations, if needed.
- Add 1.0 ul of the cell suspension (template DNA) to a final volume of 25 ul. Vortex to mix.
- Run PCR amplification.
- Hot-start: 95°C - 1 min
- 30 cycles
- Denaturation: 95°C - 30 s
- Annealing: x°C - 30 s (Adjust temperature according to primers)
- Elongation: 72°C - Calculate from expected sequence length, x kb/min
- Elongation: 72°C - 10 min
- Analyze PCR products by agarose gel electrophoresis.
Site-directed mutagenesis
Based on the QuikChange® Site-Directed Mutagenesis Kit
- Design two complimentary oligonucleotides containing the desired mutation with https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&SubPageType=ToolQCPD&PageID=15
- Prepare PCR (amounts for 100 µl reaction)
- 10 ng dsDNA template
- 125 ng primer #1
- 125 ng primer #2
- 84 µl ddH2O
- 2 µl 10 mM dNTPs
- 10 µl 10x Pfu reaction buffer
- 2 µl Pfu Turbo DNA polymerase
- Run PCR
- 95 °C - 30 sec
- 22 cycles of
- 95 °C - 30 sec
- 55 °C - 1 min
- 68 °C - 1 min/kb plasmid
- 4 °C - ∞
- Make sure the reaction is ≤ 37 °C before proceeding
- Add 2 µl (for 100 µl reaction) of Dpn I restriction enzyme and mix thoroughly
- Spin down for 1 min in microcentrifuge
- Immediately incubate at 37 °C for 1 hour or more (more gives less background)