6.4.1 Isolation of Plasmid DNA from E.coli
For the plasmid preparation the GenElute HP plasmid minipreparation kit from SIGMA-ALDRICH was used. The cells were resuspended by mixing them in 200 μL re-suspension solution. Afterwards 200 μL of lysis buffer was added and the mixture was inverted 8 times. The suspension was cleared by leaving it to stay for 5 min and 350 μL of neutralization and binding buffer was added. The mix was inverted 6 times and centrifuged in an EPPENDORF 5418 centrifuge at 14 000 rpm for 10 min. If the so-lution was not cleared, the mixture was centrifuged again at 14 000 rpm for 10 min.
The column was equilibrated by adding 500 μL of column preparation solution and centrifugation in an EPPENDORF mini spin plus centrifuge at 14 000 rpm for 1 min.
Thereafter the cleared lysate was transferred to the column and centrifuged in an EPPENDORF 5418 centrifuge at 14 000 rpm for 1 min. The flow-through liquid was dis-carded. The plasmid DNA was washed to remove contaminants by adding wash so-lution 1 and centrifuging it in an EPPENDORF 5418 centrifuge at 14 000 rpm for 1 min.
The flow-through liquid was discarded and the same procedure was done with wash solution 2. After that the DNA sample was centrifuged again in an EPPENDORF 5418 centrifuge at 14 000 rpm for 1 min and left at room temperature for 2 min to remove the excess of ethanol. After the washing and drying the plasmid DNA was eluted by adding 30 μL of water and centrifugation at 12 000 rpm for 1 min. The purification is checked by measuring concentration and absorption coefficients. A260/280 should be about 1.8, A260/230 should be about 2.0-2.2. If the values are differing, it may indi-cate the presence of proteins or phenols, respectively.
6.4.2 Agarose Gel Electrophoresis
Agarose gel electrophoresis is a method to separate DNA fragments depending on their base pair length. DNA reacts as a negative charged particle because of its phos-phate backbone. When an electronic field is applied to agarose gel the DNA frag-ments migrate through it with different speed depending on their base pair length.
The migration of the sample through agarose is also a speed defining parameter as well as the applied voltage. Smaller DNA fragments tend to migrate faster than taller ones. A marker with fragments of known base pair length is used to identify the length of the sample. To make the DNA fragments visible, a fluorescent dye is used either together with the DNA sample or in the agarose gel.
For the setup, 1% agarose in TAE buffer has to be prepared and heated up in a mi-crowave until all agarose is solved. After it is cooled to 60 °C, 3 µl/100 ml of midori green dye can be added that interacts with the DNA. Pour the solution into the mould, without generating bubbles. After casting, chill the gel for 15 to 30 min to complete gelation. The DNA samples have to be mixed with 6 x DNA sample buffer (NEB) which consists of glycerol and bromophenol. This will increase the density of the sample so it falls into the well of the gel and provides a visible marker to monitor the progress of electrophoresis. A voltage is applied until the running front is at the end of the gel. The DNA bands can then be monitored with UV-light.
6.4.3 Preparation of Electrocompetent E. coli
Electrocompetent E. coli were prepared as described early by INOUE et al..[338] Briefly, 5 mL LB preculture E. coli BL21 (DE3) ΔnemA without antibiotic were inoculated from a glycerol stock and incubated overnight at 37 °C, 200 rpm (HT INFORS shak-er). In the next morning, a 2 L baffled flask with 300 mL LB-Media was inoculated with 3 mL preculture. The flask was incubated in the shaker with 200 rpm, 37 °C. At the point the OD600 reached 0.4-0.6, the flask was cooled on ice for 30 min. Then the
cell suspension was transferred into precooled 50 mL reaction tubes and centrifuged at 4000 rpm, 4 °C for 8 min. The supernatant was discarded and the pellets washed and resuspended in several steps, using 45, 25, 2 and 0.5 mL chilled 10% glycerol.
After every resuspension step, the cells were pelleted at 4 °C and 4000 rpm for 8 min.
Finally, all cells were pooled together, centrifuged and resuspended in 1.7 mL 10%
glycerol. The cells were aliquoted in 50 µL, shock frozen in liquid nitrogen and kept frozen at -80 °C until further usage.
6.4.4 Transformation of Electrocompetent E.coli
During a transformation, exogenous DNA is taken up through the cell membrane and incorporated in the competent cell. Since E. coli does not have natural compe-tence it has to be made competent for DNA uptake. The transformation proceeds through an electromagnetic pulse. Before the pulse, the positive and negative charged particles are allocated counterbalanced. During the pulse an electromagnetic field is established and an electric potential is been induced across the cell membrane which is able to increase the cell permeability and as a consequence the cells are able to take up the exogenous DNA.
The transformation was carried out by adding 1 μL (3 μL) of digested and dialyzed DNA to 25 μL (50 μL) of competent cells on ice. The mixture was transferred to an electroporation cuvette with 2 mm gap and the BIO-RAD micropulser with the set-tings EC2 was used with typical durations for the electro transformation of 5.8-6.0 ms. Immediately 1 mL of LB-medium was added and the mixture was incu-bated in an EPPENDORF Thermomixer comfort at 37 °C and 700 rpm for 60 min. Then the cells were centrifuged and resuspended in 100 µl LB-medium. The suspension was plated on LB-Agar-antibiotic plates and the plates were incubated over night at 37 °C. The plates were then kept at 4 °C.