IGEM:Paris Bettencourt 2012/Protocols/MAGEwoR
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MAGE without Robot or Making electrocompetent cells and transforming with ssDNA
Prepare bacterial cultures
- Inoculate EcNR2 strain from frozen glycerol stock or a single colony into 3 to 5 ml LB medium. Shake at 30° to 32°C overnight.
- Add ~0.5 ml of the overnight culture to 35 ml of LB medium in a 250-ml (baffled) Erlenmeyer flask.
- Place the flask in the 32°C shaking water bath and grow cells at 32°C with shaking for ~2 hr.
- The cells are ready when the A600 is between 0.4 and 0.6. It is important not to over-grow the cells, since stationary phase cells do not express the recombination functions well.
Induce recombination functions
- Transfer half the culture to a 125-ml (baffled) Erlenmeyer flask and place that flask in the 42°C water bath. Shake 15 min at 220 rpm to induce. Leave the remainder of the culture at 32°C; this will be used as the uninduced control that lacks recombination activity. While the cells are inducing, fill an ice bucket with an ice-water slurry.
- Immediately after inducing for 15 min at 42°C, rapidly cool the flask in the icewater slurry with gentle swirling. Leave on ice for ≥5 min. Follow the same cooling protocol with the uninduced 32°C culture. While the cells are on ice, precool the centrifuge to 4°C and chill the necessary number of 35- to 50-ml plastic centrifuge tubes, labeled for induced and uninduced cells.
Make electrocompetent cells
- Transfer both the induced and uninduced cultures to the appropriately labeled chilled 35- to 50-ml centrifuge tubes. Centrifuge 7 min at 4600g (6700 rpm in a Sorvall SA-600 rotor), 4°C. Aspirate or pour off supernatant.
- Add 1 ml ice-cold distilled water to the cell pellet in the bottom of each tube and gently resuspend cells with a large pipet tip (do not vortex). Add another 30 ml ice-cold distilled water to each tube, seal, and gently invert to mix, again without vortexing. Centrifuge tubes again as in last step
- All subsequent resuspensions of cells should be done gently and without vortexing. Preparation of the cells for electroporation washes out any added chemical inducing agent.
- Decant the 30-ml supernatant very carefully from the soft pellet in each tube and resuspend each cell pellet in 1 ml ice-cold distilled water.
- Remove tubes from the centrifuge promptly. The pellet is very soft and care should be taken not to dislodge it, especially when processing multiple tubes.
- Transfer resuspended cells to microcentrifuge tubes. Microcentrifuge 30 to 60 sec at maximum speed, 4°C. Carefully aspirate supernatant. In each of the tubes, resuspend the cell pellet in 200 μl cold distilled water, which will provide enough material for four or five electroporations.
- Electrocompetent cells can be stored at −80°C after resuspending the cell pellet in 15% (v/v) glycerol. For highest efficiency, use freshly processed cells.
Introduce DNA by electroporation
- Chill the desired number of 0.1-cm electroporation cuvettes on ice. Turn on the electroporator and set to 1.80 kV.
- In microcentrifuge tubes on ice, 100 ng of single-stranded oligonucleotide with 50 to 100 μl of the suspension of induced or uninduced cells. Do the mixing and subsequent electroporation rapidly; do not leave the DNA-cell mixes on ice for extended periods. Be sure to include the following electroporation reactions and controls:
- Induced cells plus DNA.
- This is the culture that should yield the designed recombinants.
- Induced cells without DNA.
- This is a control to identify contamination, determine the reversion frequency, and obtain some idea of the efficiency of the selection.
- Uninduced cells plus DNA.
- This control tells whether there is some contaminating factor in the DNA that is contributing to the selected colonies.
- Induced cells plus DNA.
- Introduce the DNA into the cells by electroporation.
- The time constant should be greater than 5 msec for optimal results. Low time constants indicate problems with the cells, the DNA, or even the equipment.
- Immediately after electroporation, add 1 ml LB medium to the cuvette using a micropipettor with a 1000-μl pipet tip. Transfer the electroporation mix to sterile culture tubes and incubate the tubes with shaking at 30° to 34°C for 30min to 2 hr
Determine cell titers
- Make serial 1:10 dilutions of the electroporation mix through 10^−6 using M9medium or 1× TM buffer, dispensing 0.9 ml M9 or TM and 0.1 ml of the cell suspension per tube.
- The dilutions can be made in rich medium if a selection for antibiotic resistance is applied.
- To determine total viable cell count, spread 100 μl of 10^−5 and 10^−6 dilutions on LB plates (rich plates without drug). Incubate the plates at 30° to 34°C for 1 to 2 days.
- To determine recombinant cell count, plate cells on selective plates as follows depending on the anticipated recombinant frequency.
- If efficient recombination is expected, spread both 10 and 100 μl of the 10^−1 and 10^−2 dilutions.
- If low numbers of recombinants are expected, spread 100 μl each of a 1:5 and 1:10 dilution.
- For the no-DNA and uninduced controls, plate 200 μl directly on selective plates.
- Incubate plates at the appropriate temperature (30° to 34°C).
- At 30°C, colonies may take two days to come up on LB plates.
- Candidates should be purified by streaking for single colonies and retested for the appropriate phenotype.