MgCl2 (100X)	Volume	Notes
MgCl2	20.3 g	Sterilize
H2O	Until 100mL	Sterilize

3. Measuring OD

C _{0 1 x V0 1=C x V V=20 mL C=0.2 g/mol}

_{Vo=(0.2 X 20)/(0,38 X 5)=2.11 mL of culture is necessary to obtain the required optical density.}

_{E. coli Transformation}

_{Electroporation method was used in order to transform a DNA construction. This procedure is commonly used for E. coli and Agrobacterium.}

_{1.The electroporation cuvette was put in ice 10 minutes before inserting the cells. An aliquot of electrocompetent cells are taken out of the -80°C freezer, and they are put immediately into ice.}

_{2.1-2 ul of the ligation product are taken and added carefully to them.}

_{3.60 ul of the mix are taken and put into an electroporation cuvette making sure that there are no bubbles.}

_{4.The cuvette is dried and put in the electroporator, making sure that any spark is done. In that case, the process does not work and must be repeated. This could happen when plasmid and E. coli concentration are not optimal.}

_{5.The voltage for the electroporator is 1500V for E. coli and 1440V for Agrobacterium.}

_{6.Transformed cells are resuspended in 300 μL of LB in the electroporation cuvette. Then, they are taken and put into an Eppendorf letting them grow in the shaker for 2 hours at 37°C.}

_Minipreps

_{In order to carry out all of necessary Miniprep -extraction of the plasmids out of E. coli -the protocol of the Omega kit (Plasmid DNA Mini Kit I Spin Protocol) is used. The steps to do it are:}

_{1.Grow 1-5 mL culture overnight in a 10-20 mL culture tube.}

_{2.Centrifuge at 10.000 x g for 1minute at room temperature. Decant or aspirate and discard the culture media.}

_{3.Add 250 ul Solution I mixed with RNase A. Vortex or pipet up and down to mix thoroughly. Transfer suspension into a new 1.5mL microcentrifuge tube.}

_{4.Add 250 ul Solutions II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary. Avoid vigorous mixing and do not exceed a 5 minute incubation.}

_{5.Add 350 ul Solution III. Immediately invert several times until a flocculent white precipitate forms. Centrifuge at maximum speed (>13.000xg) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.}

_{6.Insert a HiBind DNA Mini Column into a 2 mL Collection tube.}

_{7.Transfer the cleared supernatant from Step 6 CAREFULLY aspirating it into the HiBind DNA Mini Column. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.}

_{8.Add 500 ul HBC Buffer diluted with isopropanol}

_{9.Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse collection tube.}

_{10.Add 700 ul DNA Wash Buffer diluted with ethanol. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.}

_{11.Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the column. This step is critical for removal of trace ethanol that may interfere with downstream applications.}

_{12.Transfer the HiBind DNA Mini Column into a nuclease-free 1.5 mL microcentrifuge tube.}

_{13.Add 50 ul Elution Buffer or sterile deionized water directly to the center of the column membrane.Let sit at room temperature for 1 minute. Centrifuge at maximum speed for 1 minute.}

_{14.Store eluted DNA at -20°C.}

_{Petri dish culture}

_{Bacteria are transformed with a plasmid which gives them resistance to a specific antibiotic. Petri dishes must include this antibiotic to reject those bacteria that have not acquired the plasmid.}

• _{E. coli- pUPD2 plasmids: chloramphenicol.}
• _{E. coli- Alpha 1 and 2: kanamycin.}
• _{E. coli- Omega 1 and 2: streptomycin}
• _{Agrobacterium: rifampicin + the specific one for each construction.}

_{To carry out this procedure is necessary to work in the laminar flux cabinet.}

_{This procedure is necessary to made it in the laminar flux cabinet. After E. coli transformation, cells are put into an Eppendorf letting them grow at 37°C. The spread plate method is done with 50-40 ul of this bacteria culture. To plate it is used aIt is used for plating a glass dipstick. After that, E. coli culture plates are put at 37°C for approximately 16h. Agrobacterium growth needs 32h at 28°C.}

_{Ligation reaction}

_{The assembly of DNA fragments, commonly referred to as Golden Gate assembly, is an efficient technique where multiple inserts could be assembled into a vector backbone. The net result is the ordered and seamless assembly of DNA fragments in only one reaction. The ligations have a total volume of 10 μL so all of sequences that form part of the constructions are mixed up in an Eppendorf of 0.2mL.}

_{This mix is put in a thermocycler with the programs GB (Golden Braid) or GG (Golden Gate).}