Difference between revisions of "Team:Vilnius-Lithuania/Methods"

Inoculate LB medium with 1 mL of JM107 bacteria. Inoculate 500 mL of LB medium with 500 µL of JM107 bacteria and grow until OD₆₀₀ of 0.5–0.7. Once the bacteria has reached the desired OD, they are taken out of incubation and put on ice for 15 minutes. Starting from this moment, all the steps are done on ice or in a cold temperature (4ºC). Tube is centifuged for half an hour in 3000 G and washed with distilled autoclaved water. Afterwards, they are once again centifuged for half an hour in 3000 G. This process (washing and centrifuging) is repeated for 5 times. After the last spin, bacteria is washed with glycerol solution. 100 µL of aliquot is pipetted into the tubes and ready for transformation.

Turn on electroporator and optimize it for used bacteria strain. Place 1 mm electroporation cuvettes on ice. Add 1 µL of ligate/plasmid DNA solution to the cells in each of the microcentrifuge tubes. Transfer this mixture to the cold cuvette, tap on countertop two times, wipe water from exterior of cuvette and place in the electroporation module and press pulse. Add 1 mL of 37ºC LB medium, mix up by pipetting up and down and transfer to a 15 mL falcon tube. Shake in the incubator for one hour. Finally, place 10 µL of mixture on one LB-antibiotic plate, 100 µL on another and 200 µL on third and incubate them overnight at 37ºC.

All the reaction components are mixed on ice in specific proportions (adding polymerase last) and quickly transferred into preheated thermocycler.

All the reaction components, except the bacteria, are mixed on ice in specific proportions (adding polymerase last). To each PCR tube, containing the PCR reaction, the small amount of bacteria from colony is added. This is made with a wooden stick and each colony is marked on the plate, as well as transferred onto a new plate. The PCR tubes are quickly transferred into a preheated thermocycler.

All the reaction components are mixed on ice, adding the restriction enzymes last. Tubes are incubated at 37ºC for half an hour.

All the reaction components are mixed on ice, adding the ligase enzyme last. Tubes are incubated at room temperature for 10-15 minutes. Then ligase enzyme is inactivated by incubating tubes at 65ºC for 10 minutes. Afterwards, ligation reaction is chilled on ice and used in transformation.