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.