Bacterial Techniques

Electrocompetent bacteria (DH10B, Life technologies) were used for the electroporation. A 100 Hl aliquot of frozen bacteria (from -80°C) was thawed on ice for 10 minutes and 25 Hl added to each ligation reaction (1 ng plasmid DNA) in a volume of 15 Hl and mixed. The bacteria-DNA mix was then transferred to a precooled electroporation cuvette (Bio-Rad, electrode gap 0.1 cm) and electroporated (1.8 kV, 2. X, 25HF):(Fig. 2.6). The time constant was normally between 4.5 and 5.5 ms. The bacteria were then immediately resuspended in 1ml SOC medium (2% bactotryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 20 mM glucose in sterile milli-Q water pH 7.5) and transferred to a 15 ml glass test tube. The transformation mix was incubated in a shaker at 220 rpm (MultitronR, AJ110/111) for 1 hour at 37°C. 50-100 Hl of the transformation mix was then plated out on agar plates (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% bacto agar in sterile milli-Q water) containing the appropriate antibiotic (50 Hg/ml ampicillin, 30 Hg/ml kanamycin or 25 Hg/ml tetracyline) and incubated at 37°C overnight.

2.3.2 Preparation of Plasmid DNA

2.3.2.1 Small Scale Preparation of Plasmid DNA (QIAscreen)

In order to analyse a bacterial colony growing on an agar plate after electroporation, plasmid DNA was isolated by the QIAscreen method (Qiagen). 2-10 Hg plasmid DNA per colony picked could be isolated using this technique, depending on the copy number of the plasmid (pBR322 derivative 15-200 copies/cell, pUC derivative 500-700 copies/cell). This amount is usually enough to perform restriction digests to check the DNA isolated.

2ml of Luria-Bertani (LB)-Broth (1% bactotryptone, 1% NaCl, 0.5% yeast extract) containing antibiotic in a 15 ml tube were inoculated with a single bacterial colony and incubated in a shaker (220 rpm) at 37°C overnight. A 1.5 ml aliquot of the bacterial suspension was then transferred to a 1.5 ml eppendorf tube and the bacteria pelleted in a tabletop centrifuge (6000 x g) for 5 minutes at room temperature. The pellet was resuspended in 300 Hl buffer P1 (50 mM Tris/HCl, 10 mM EDTA, 100 Hg/ml RNase A). 300 Hl buffer P2 (200 mM NaOH, 1% SDS) was added and the suspension mixed by gentle inversion of the reaction tube. After an incubation period of 5 minutes at room temperature in which the bacterial membranes were lysed by buffer 2, 300 Hl buffer P3 (3 M KAc pH 5.5) was added to precipitate the bacterial genomic DNA and proteins. The reaction tubes were inverted several times and the mixture centrifuged in a tabletop centrifuge (10000 x g) for 20 minutes. The supernatant, containing the plasmid DNA, was transferred into a new reaction tube, the DNA precipitated with 0.8 volumes of isopropanol and centrifuged (10000 x g) at room temperature for 20 minutes. The DNA pellet was then washed once with 500 Hl ethanol (70%), centrifuged once more for 20 minutes (10000 x g) and dried in a vacuum dryer. The dried pellet was resuspended in 40 Hl H₂O and DNA analysed by restriction digestion. For a digest 8 Hl of plasmid solution was normally used in a total volume of 10 Hl. 2.3.2.2 Large Scale Preparation of Plasmid DNA (Qiagen maxi prep)

A large scale plasmid DNA preparation was made by inoculating 200 ml of LB-broth including antibiotic with 100 Hl of bacterial suspension from a small scale plasmid preparation. The bacteria were incubated at 37°C in a shaker (220 rpm) overnight. 10 ml of the bacterial suspension was taken for freezing (see 2.3.3), and the rest of the suspension (190 ml) was centrifuged for 10 minutes (Beckman, JLA-16.250, 5524 x g) at 4°C. The supernatant was discarded and the pellet resuspended in 10 ml ice cold buffer P1 (50 mM Tris/HCl, 10 mM EDTA, 100 Hg/ml RNase A, pH 8) and transferred to a 40 ml tube. To lyse the bacterial membranes, 10 ml of buffer P2 (200 mM NaOH, 1% SDS) was then added and the components carefully mixed by inversion of the tube. After incubation for 5 minutes at room temperature, 10 ml of buffer P3 (3 M KAc pH 5.5) was added and gently mixed again. The mixture was incubated for 20 minutes on ice, and was then centrifuged (Beckmann, JA-25.50, 30966 x g) for 30 minutes at 4°C. The supernatant was decanted into a new 40 ml tube and centrifuged again for 15 minutes to separate residual bacterial proteins and precipitated SDS from the plasmid DNA. The supernatant was then applied to an ion exchange column (Qiagen- tip 500) that had been previously equilibrated with 10 ml of buffer QBT (750 mM NaCl, 50 mM MOPS, 15% ethanol, 0.15% Triton X-100). The detergent present (Triton X-100) in this buffer lowers the surface tension of the buffer so that it enters the column by gravity flow. After the supernatant had passed through the column, it was washed two times with 25 ml of buffer QC (1 M NaCl, 50

mM MOPS pH 7.0, 15% ethanol). Under these conditions (1 M NaCl) RNA and proteins are washed from the column whereas the plasmid DNA remains bound. The DNA was then eluted (because of high salt conditions) into a new 40 ml tube with 15 ml of buffer QF (1.25 M NaCl, 50 mM MOPS pH 8.5, 15% ethanol). The plasmid DNA was then precipitated by the addition of 0.8 volume of isopropanol and incubated at room temperature for 5 minutes before being centrifuged (Beckman, JS.13.1, 26688 x g) for 30 minutes at 4°C. The pellet was washed with 5 ml ethanol (70%) and after centrifuging once more for 15 minutes, dried in a vacuum dryer. The pellet was resuspended in 400 Hl of 1 mM EDTA and left to dissolve overnight at 4°C. The concentration was then determined (as described in 2.2.3) and the DNA examined by digestion with restrictions enzymes.

2.3.3 Freezing of Transformed Bacteria

For each large scale preparation, 10 ml of the overnight culture was transferred to a 40 ml tube and centrifuged (Beckman, JA 25.50, 4355 x g) for 5 minutes at 4°C. The supernatant was discarded and the pellet resuspended in 1.8 ml of a freezing medium (95% LB-broth, 5% DMSO). The bacterial suspension was then stored at -20°C. Addition of the DMSO inhibits formation of ice crystals during the freezing and thawing process.

2.4 Cell Culture Methods