Team:DTU-Denmark/Methods/Protocols

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Protocols

Glycerol stock-preservation

Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.
The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.
Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.
Mark the PCR tubes on lid and side and place in thermocycler.
The annealing temperature should match the primer melting temperatures minus 5 degrees. This can be hard since Tm predictors tend to disagree.
Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).

Each reaction should contain:
- 1X NEBuffer (according to the desired restriction enzyme(s)) with BSA.
- 1 unit/μl restriction enzyme.
- The DNA that is to be cut, final concentration of 10 ng/μL
- Milli-Q water to a volume of 20 μL
Incubate the reactions at the restriction enzymes’ optimal temperature. If the restriction reaction is analytical, 30 minutes is enough. If the DNA is to be purified, a longer duration is recommended, e.g. 3 hours.
Add loading buffer to samples and run on 1% agarose gel with ethidium bromide (0.2 μg/ml).
Analyze your gel, and cut bands out if desired.

2 mL LB is inoculated with cells from a single colony and incubated at 37 °C O/N at 350 rpm
200 mL of preheated (37 °C) LB is inoculated with 10 ul culture from step 1 in a shake flask and incubated at 37 °C and 350 rpm until OD 600 reaches 0.3 - 0.6
When OD600 reaches 0.3 - 0.6 the shake flask is chilled in an ice-bath until the culture is completely cooled. All steps from here should be kept as cold as possible.
The 200 mL culture is transferred to 50 mL tubes and spun at 5500 G for 10 minutes. The supernatants are discarded and each pellet is resuspended in 25 mL ice cold 0.1 Molar CaCl₂. The tubes are poured together to 2x50 mL.
The cells are spun at 5500 G for 10 minutes and the supernatant is discarded. The cells are resuspended in 1 mL 0.1 Molar CaCl₂ and transferred to microcentrifuge tubes.
The cells are spun at 15000 G for 2 minutes and the supernatant is discarded. The cells are resuspended in 1 ml 0.1 Molar CaCl2 with 15% glycerol.
The cells are aliquoted into portions of 200 μL in pre-chilled Eppendorf tubes and immediately stored at -80 °C.

Transformation with chemically competent E. coli IMPORTANT: Keep everything on ice unless otherwise stated in the protocol.

Competent cells are defrosted in an ice bath
50 μL cells is transferred to a cooled eppendorf tube
1-5 μl DNA is transferred to the competent cells, mixed by gently swirling the tube
DNA and cell mix is kept on ice for 10 minutes
Cells are heat shocked at 42 °C for 30 seconds in water bath
Cells are kept on ice for 3 minutes.
500 μL LB without antibiotics is added and incubated at 37 °C for 30-120 minutes depending on antibiotic marker
Plate cells on LB plate with the relevant antibiotic and incubate overnight at 37 °C

IMPORTANT.! Keep everything on ice unless otherwise stated in protocol.

Preparation: Put electro cuvettes, LB media with no antibiotics and DNA on ice in order to cool it down.

Defrost electro-competent cells on ice
Transfer 1 μl DNA to the competent cells
Transfer DNA and cell mix to electroporation cuvette
When ready the cuvette is quickly dried with paper towel and inserted to the electroporater and a shock is given. Immediately after the shock is given transfer 1 ml of ice cold LB to the cuvette and transfer the cells and media from the cuvette to an eppendorf tube and put the tube on ice.
Leave the cells on ice for 10 minutes.
Incubate the cells at 37 °C for 30-60 minutes depending on antibiotic marker
Plate 100 μl on a half plate of LB with required antibiotic
Spin the cells briefly at maximum RPM and decant the supernatant gently, approximately 100 μl will remain in the tube, resuspend the pellet in the rest of the supernatant and plate it on the other half of the plate used in step 7.
Incubate plates at 37 °C overnight