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 manufacturer'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:

Competent cells are centrifuged (2 min., 6000 rpm, 4 ºC). Supernatant is discarded, leaving about 100 µL of solution, in which cells are re-suspended. These cells are mixed with 10-20 ng of ligate or plasmid DNA. The mixture is incubated on ice for half an hour, then placed in a thermostat at 42 ºC for 1 minute. 1 mL of LB liquid medium is added to the mixture and cells are incubated in a shaking thermostat for 1 hour at 37 ºC. The tubes are pelleted by centrifuging; supernatant is discarded, leaving about 50 µL, in which cells are re-suspended. Transformants are placed onto a Petri dish with LB medium and strain-specific/vector-specific antibiotic. Plates are incubated in a thermostat at 37 ºC for 16 hours.

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:
Bacteria cells
LB medium
Antibiotic
IPTG (1mM)
Arabinose (0.2%)
PMSF (peptidase inhibitor)
Tris-HCl + 0.1 M NaCl buffer (pH=8)
1mL 0.1 M NaOH
1 % SDS solution

Process:

Transformed bacteria are grown overnight at 37 ºC in a 4 mL of LB medium and antibiotic. Next day, the overnight culture is transferred to the tubes with 20 mL LB medium at a dilution 1:20 and grown for 3 hours at 37 ºC. 0.2% arabinose is added to each tube. Bacteria are then grown at 37 ºC for 4-5 hours. Cells are pelleted and stored in -20 ºC or re-suspended in 1 mL Tris-NaCl buffer. 10 µL of PSMF is added to each tube. Cells are then sonicated and centrifuged for 20 minutes at 13200 rpm. Soluble fraction is transferred to the other eppendorf tube, while insoluble fraction is suspended in 1 mL of NaOH-SDS solution.

Materials:
Acrylamide solution
Tris-HCl (pH 8.8) buffer
Tris-HCl (pH 6.8) buffer
Amonium persulphate solution (12 %)
TEMED solution
Milli-Q H₂O
Tris-Gly-SDS buffer
Protein loading dye
“Page Blue” gel dye
Protein samples
Protein ladder

Process:

Two 12 % SDS protein electrophoresis gels are made - one for western bloting and one for SDS-PAGE. Each gel consists of separating (lower) and stacking (upper) gels. For the separating gel acrylamide solution, Tris-HCl (pH 8,8) buffer and milli-Q H2O is mixed with x μL APS and x μL TEMED solutions and poured into the gel preparation frame with the water on top of it. After that the stacking gel is made from acrylamide solution, Tris-HCl (pH 6,8) buffer, milli-Q H2O, x μL of APS and x μL of TEMED. Whole mixture is poured into the gel preparation frame (on top of the separating gel), comb inserted into the stacking gel and everything is left to gelate. Protein sample preparation: 45 μL of soluble and insoluble fraction samples after lysation are mixed together with 15 μL loading protein dye and denaturated for 10 minutes at 100°C. Prepared gel is put into gel loading machine, samples are loaded into the wells, as well as protein ladder. Reaction conditions are x V/cm voltage, gel is loaded for x minutes. After electrophoresis, one gel is placed in the transfer buffer, while the other is dyed with Page Blue dye.

Materials:
PBS buffer
PBS-T buffer
TBS buffer
PTB buffer
Blocking buffer
Protein Ladder
PVDF membraine
Whatman paper

Process:

Load SDS-PAGE, but do not dye the gel. The membraine and two pieces of Whatman paper are prepared: membraine is put into EtOH for 15 s, then put into PTB buffer; papers are put only in PTB buffer for 10 minutes. Polyacrilamid gel is also put into PBS buffer for 10 minutes.

Then “sandwich” is made on a blotting machine: firstly goes one Whatman paper, then membraine, on top of the membraine goes the gel, and on top of a gel goes second Whatman paper. Transfer is performed for 40 minutes (11 V). After transfer, membraine is blocked for 1 hour in the blocking buffer. Afterwards, membraine is washed with PBS-T buffer and placed into 50 mL cylinder. 3 mL of PBS-T buffer and primary antibodies are also placed in the cylinder. Incubation overnight.

After incubation, membraine is washed again with PBS-T buffer in following order: half of the cilinder is filled with PBS-T buffer and washed for 15 minutes, then buffer is changed and membraine is washed with fresh PBS-T buffer twice for 5 minutes. After washing, secondary antibodies and 3 mL of PBS-T buffer are again placed into the cylinder and incubated for 2 hours.

After incubation, membraine wash is repeated exactly as before, only the last time membraine is washed with TBS buffer instead of PTS-T. After washing visualizing solution is made: 1 tablet of Chlornaftol is dilluted in 10 mL of EtOH; 10 mL of TBS buffer, 2 mL of Chlornaftol solution and 15 µl of H2O2 are placed into the visualizing bath. Membraine is put into the prepared solution. After the signal becomes visible, membraine is placed into water.

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.