Team:Vilnius-Lithuania/Methods
4 mL of LB medium
Antibiotic
Transformed bacteria
Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacterer's instructions (pdf file).
Agarose gel
DNA ladder
6x Loading Dye
1x TAE buffer
The agarose gel is loaded into electrophoresis sytem, filled with the buffer. The DNA ladder and DNA samples are then loaded into the wells. Run the gel at 130V for 30-40 minutes.
Competent cells
Ligate/plasmid DNA
LB medium
Plates with LB medium and antibiotic
In order to perform transformation, the prepared competent cells are centrifuged (2 minutes, 6000 rpm, 4ºC) and most of the supernantant is discarded, leaving about 100 μL of solution, in which cells are suspended.
These cells are mixed with 10-20 ng of ligate or plasmid DNA. The mixture is incubated on ice for half an hour and right after the eppendorf tubes are placed in a thermostat in 42ºC for 1 minute. This is why it is called heat-schock transformation.
1 mL of LB liquid medium (without antibiotic) is added to the eppendorf tube with the mixture and grown in a shaking thermostat for an hour at 37ºC. After the tubes are pelleted by centrifuging, supernantant is discarded, leaving about 100 μL, in which cells are suspended.
Transformants are placed onto a Petri dish with LB medium and specific antibiotic. Plates are incubated in a thermostat at 37ºC for a night.
LB medium
NaCl solution
CaCl2 solution
Bacterial cells
Grow 4 mL LB medium with the cells for 2-3 hours at 37ºC in the shaker. The cells are grown to OD600 of 0.4-0.5. All the further steps are done on ice. Firstly, the cells are transferred into eppendorf tubes and pelleted in a centrifuge for 2 minutes at 6000 rpm at 4ºC. Supernatant is discarded and bacterial cells are suspended in 1 mL of NaCl solution. Tubes are centifuged once again for 2 minutes, 6000 rpm, 4ºC. Supernantant is discarded and cells are resuspended in 1 mL of CaCl2 solution. They are left on ice for an hour or longer.
BL21(DE3) bacteria cells
LB medium
specific antibiotic
IPTG (1M)
Transform plasmid DNA into BL21 (DE3) bacteria and grow overnight at 37ºC on a Petri dish with specific antibiotic. The next day inoculate bacteria to 4 mL of LB medium and grow overnight. On the third day, transfer the night culture into a few tubes with 20 mL LB medium at a dilution 1:50 and grow for 3 hours in 37ºC (until OD600 ~ 0.6). Add 20 μL of IPTG (1M) into each tube. Then half of bacteria are incubated for 3 h in 37ºC, the other half is incubated for 16 h in 16ºC.