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 manufacturer's instructions.
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 (1mM)
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.
Acrylamide solution
Tris-HCl (pH 8.8) buffer
Amonium persulphate solution
TEMED solution
Milli H2O
Tris-Gly-SDS buffer
Protein dye
“Page Blue” gel dye
Protein samples
Protein ladder
Distributive solution is made (6 mL of acrylamide solution with 3.75 mL of Tris-HCl buffer and 5.25 mL of water). Then fast-freezing solution is made: 2 mL of distributive solution is mixed with 50 μL of amonium persulphate solution and 5 μL of TEMED solution, and poured into the gel preparation frame. Freeze for 5 minutes.
After, 50 μL of amonium persulphate and 10 μL of TEMED solution are poured into residual distributive solution. Whole mixture is poured into the gel preparation frame (on top of fast-freezing gel). Water is added on top. Freeze for 40 minutes.
Focusing gel is made: 0.65 mL of acrylamide solution, 1.25 mL of Tris-HCl, 3.05 mL of water, 25 μL of amonium persulphate and 5 μL of TEMED. After distributive gel fully freezes, focusing gel is poured on top of it, comb inserted and whole gel is left to freeze for additional 30 minutes. After gel is fully freezed, gel making frame is dismanteled and gel is rinsed with water.
Protein preparation: proteins are mixed together with protein dye and denaturated 10 minutes at 100°C. Prepared gel is put into gel loading machine and poured with Tris-Gly-NDS buffer. Then samples are loaded into the wells. Reaction conditions are 15 V/cm voltage, gel is loaded for 40 minutes. After SDS-PAGE, gel is dyed with “Page Blue” dye, according to manufacturer's recommendations.
PBS buffer
PBS-T buffer
TBS buffer
PTB buffer
Blocking buffer
Protein Ladder
PVDF membraine
Whatman paper
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.
JM107 bacteria
LB medium
Milli-Q H2O
Glycerol solution (1/2 glycerol, 1/2 H2O)
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.
Electroporator
Electro-competent cells
Plates with LB medium and specific antibiotic
Ligate/plasmid DNA
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.
DreamTaq polymerase
Long PCR polymerase
Phusion polymerase
Primers
10x PCR buffer
dNTP
Milli-Q H2O
DNA
All the reaction components are mixed on ice in specific proportions (adding polymerase last) and quickly transferred into preheated thermocycler.
DreamTaq polymerase
Phusion polymerase
dNTP
Primers
10x PCR buffer
Milli-Q H2O
Bacteria from colony
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.
Restriction enzymes
10x Fast Digest buffer
FastAP
DNA
Milli-Q H2O
All the reaction components are mixed on ice, adding the restriction enzymes last. Tubes are incubated at 37ºC for half an hour.
T4 Ligase
10x T4 Ligase buffer
Vector DNA
Plasmid DNA
Milli-Q H2O
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.