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2.6.1.1 Small-scale preparation of plasmid DNA from bacteria

For routine use, plasmid DNA was isolated from E. coli using a modified alkaline lysis procedure (Sambrook et al., 1989). A 1.5 ml aliquot of a 10 ml overnight culture was harvested by centrifugation (12,500 x g, 3 min). After removing the supernatant by aspiration, the pellet was resuspended in 100 μl of Solution I (25 mM Tris-HCl pH 7.5, 10 mM EDTA, and 15% sucrose) and incubated on ice for 5 min. Cells were lysed by adding 200 μl of Solution II (0.2 M NaOH, 1% SDS) followed by gentle inversion and incubating on ice for a further 5 min. Then, 150 μl of Solution III (3 M sodium acetate, pH 4.8) was added to the mixture, and following gentle inversion the mix was incubated on ice for 5 min. After centrifugation (12,500 x g, 15 min, 4oC), the resultant supernatant was extracted with an equal volume of phenol/chloroform (1:1) and centrifuged (12,500 x g, 5 min, 4oC). Plasmid DNA was precipitated by addition of 2 volumes of absolute ethanol and standing at room temperature for 5 min. Following centrifugation (12,500 x g, 10 min, 4oC), the pellet was washed with 70% ethanol, dried (in vacuo) using a speed Vac concentrator (Selby), resuspended in 50 μl sterile dH2O and stored at –20oC.

2.6.1.2 Genomic DNA preparation from M. elsdenii

The M. elsdenii genomic DNA was purified by the method as described by Becker et al., 1993. Frozen M. elsdenii cells (0.5 g) were ground in a mortar cooled with liquid nitrogen, and dispersed in a 10 ml solution containing 50 mM Tris-HCl, pH 8.5, 100 mM EDTA, and 200 mM NaCl. The cells were lysed by the addition of 0.5 ml of 10% SDS and 2.5 mg of proteinase K followed by incubation at 50oC for several hours.

The homogenate was extracted with an equal volume of phenol saturated with 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA (TE buffer), using a wrist action rocker for 15 min. The phases were separated by centrifuging the mixture at 3,000 rpm for 10 min at 10oC. This extraction was repeated once with a 1:1 phenol-chloroform-isoamyl alcohol) and then extraction was repeated one more time with chloroform-isoamyl alcohol alone. The aqueous phase was then adjusted to 0.3 M with sodium acetate, pH 5.2. The DNA was precipitated by the addition of 2-propanol (0.8 volume). Next, the DNA pellet was removed with a plastic pipette and transferred to 70% ethanol, spun down, dried and resuspended in 0.5 ml of TE buffer to which RNase A was added (55 μg/ml). The yield of M. elsdenii genomic DNA was approximately 1 mg of DNA/g of bacteria.

2.6.1.3 Genomic DNA preparation from Pseudomonas strains

The P. putida genomic DNA was isolated as described previously (Ausubel et al., 1994) with some modifications. A 10 ml culture of each strain was grown to saturation. The cells were centrifuged (4,000 x g, 10 min) and resuspended gently in 567 μl TE buffer. The cells were lysed by the addition of 30 μl of 10% SDS and 30 μl of 20 mg/ml proteinase K in a microcentrifuge tube, followed by incubation at 37oC for 1 h. After incubation, 100 μl of 5 M NaCl was added, mixed thoroughly and then 80 μl of CTAB/NaCl solution (4.1 g NaCl in 80 ml water and slowly add 10 g CTAB [hexadecyltrimethyl ammonium bromide] while heating and stirring, made up to 100 ml with water) added, thoroughly mixed followed by incubation at 65oC for 10 min. The homogenate was extracted with an equal volume of chloroform-isoamyl alcohol thoroughly. The phases were separated by centrifuging the mixture in a microcentrifuge for 5 min at room temperature. The aqueous phase was transferred to a fresh microcentrifuge tube and re-extracted with a 1:1 phenol-chloroform-isoamyl alcohol by mixing thoroughly, then centrifuging for 5 min. The aqueous phase was transferred to a fresh microcentrifuge tube to precipitate DNA with 0.6 volumes of isopropanol, spinning down the pellet for 5 min, and washing the DNA with 1 ml of 70% ethanol (-20oC), and centrifuged for 5 min at room temperature. The supernatant was removed and the DNA pellet was air dried and resuspended in 100 μl TE buffer.

2.6.1.4

Wizard Mini-prep for preparation of plasmid DNA from

bacteria

This method was used only when extremely pure plasmid DNA was required (for such purposes as DNA sequencing) using a commercially available kit (Promega Wizard Minipreps DNA Purification System). An aliquot (10 ml) of overnight bacterial culture was harvested by centrifugation (10,000 x g, 5 min). After removing the supernatant, the cells were resuspended by vortexing in 250 μl Cell Resuspension Solution (supplied with kit) and completely resuspend the cell pellet by vortexing well. To the suspension, 250 μl of Cell Lysis Solution (supplied with kit) was added; the microcentrifuge tube was then inverted several times to gently mix the content. After the mixture became almost clear, added 10 μl of Alkaline Protease Solution (supplied with kit) and mixed by inverting the tube four times and incubated for 5 min at room temperature. Neutralizing Solution 350 μl (supplied with kit) was added and the

contents mixed by inverting several times. The bacterial lysate was centrifuged (14,000 x g, 10 min) at room temperature. The 850 μl clear lysate was transferred into the WizardTM Plus SV Minipreps Spin Column inserted into a 2 ml collection tube and centrifuged (14,000 x g, 1 min) at room temperature. The WizardTM Plus SV Minipreps Spin Column was removed from the tube and the flow-through discarded from the collection tube. Column Wash Solution 750 μl (supplied with kit) was added to the minicolumn and centrifuged (14,000 x g, 1 min) at room temperature. Once again, the column was removed from the tube and the flow-through discarded from the collection tube and the column finally rewashed with 250 μl of Column Wash Solution by centrifuging (14,000 x g, 2 min) at room temperature. The column was transferred to a clean, sterile 1.5 ml microcentrifuge tube and the plasmid DNA finally eluted by adding 100 μl of Nuclease-Free Water (supplied with kit) to the column. The column was centrifuged at 14, 000 x g for 1 min at room temperature in a microcentrifuge tube and eluted DNA stored at –20oC.

2.6.1.5

Determination of quality and quantity of DNA by

spectrophotometry

DNA concentrations were determined using the spectrophotometric method as described by Sambrook et al., 1989. Amounts of synthesized oligonucleotides were determined using the spectrophotometric method. Isolated nucleic acid samples and synthesized oligonucleotides were diluted 1:50 (for DNA) or 1:100 (for oligonucleotides) in distilled water and absorbance readings were taken at wavelengths of 260 nm and 280 nm, and the A260/A280 ratio provided an estimation of

the purity of samples. Nucleic acid concentration was determined according to the equation: 1 A260 unit (1 cm light path) = 50 μg/ml solution for double-stranded DNA and 33 μg/ml solution for single-stranded oligonucleotides.

2.6.1.6

Quantification of DNA using the agarose plate method

Agarose plate method was used as described by Sambrook et al., 1989. A 1% agarose solution containing ethidium bromide (2 μl) was prepared in 1x TAE (Tris- acetate-EDTA) buffer (50 ml). DNA samples were prepared in sterile distilled water (5 μl) containing loading buffer (2 μl) and DNA (3 μl). The samples (10 μl) were loaded into the gel and run for one hour at 80 V with 1x TAE as the running buffer. DNA concentration standards (varying from 1-20 μg /ml) were run in parallel with the DNA samples. The quantity of DNA was estimated after photography of the gel by comparing the intensity of the sample DNA with the DNA markers.