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

Materials:
4 mL of LB medium
Antibiotic
Transformed bacteria

Process:
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.

Materials:
Agarose gel
DNA ladder
6x Loading Dye
1x TAE buffer

Process:
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.

Materials:
Competent cells
Ligate/plasmid DNA
LB medium
Plates with LB medium and antibiotic

Process:

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.

Materials:
LB medium
NaCl solution
CaCl₂ solution
Bacterial cells

Process:

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.

Materials:
BL21(DE3) bacteria cells
LB medium
specific antibiotic
IPTG (1mM)

Process:

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 (1mM) into each tube. Then half of bacteria are incubated for 3 h in 37ºC, the other half is incubated for 16 h in 16ºC.

Materials:

Process:

Materials:

Process:

Mutagenesis was performed according to manufacturer's instructions.

Gel extraction was performed according to manufacturer's instructions.

PCR purification was performed according to manufacturer's instructions.

Materials:
JM107 bacteria
LB medium
Milli-Q H₂O
Glycerol solution (1/2 glycerol, 1/2 H₂O)

Process:

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.

Materials:
Electroporator
Electro-competent cells
Plates with LB medium and specific antibiotic
Ligate/plasmid DNA

Process:

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.

Materials:
DreamTaq polymerase
Long PCR polymerase
Phusion polymerase
Primers
10x PCR buffer
dNTP
Milli-Q H₂O
DNA

Process:

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

Materials:
DreamTaq polymerase
Phusion polymerase
dNTP
Primers
10x PCR buffer
Milli-Q H₂O
Bacteria from colony

Process:

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.

Materials:
Restriction enzymes
10x Fast Digest buffer
FastAP
DNA
Milli-Q H₂O

Process:

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

Materials:
T4 Ligase
10x T4 Ligase buffer
Vector DNA
Plasmid DNA
Milli-Q H₂O

Process:

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.