Isolation of DNA

Before the isolation of plasmid, cosmid or genomic DNA, cells from cultures were harvested by centrifugation either using a miniSpin plus centrifuge/centrifuge 5415D (Eppendorf, Hamburg, Germany) or when cooling was required a centrifuge 5417R (Eppendorf, Hamburg, Germany). Higher volumes of cell cultures were pelleted using a Sorvall^® RC 6+™

centrifuge with Sorvall^® SS-34 and SLA-150 Super-Lite^® autoclavable rotors (Thermo Electron Corporation, Langenselbold, Germany).

4.1.1 Isolation of plasmid and cosmid DNA by commercial kits

Highly pure plasmids or cosmids were isolated by appliance of different purchased DNA isolation kits given in Table 8. Culture volumes of 5 mL were sufficient for isolation of plasmid DNA, in contrast cosmid DNA preparation demanded for culture volumes up to 30 mL. The application of these DNA isolation kits was done according to manufacturer’s instructions.

Cosmids were treated as low copy plasmids, the procedure for isolation of low copy vector DNA was given in the corresponding manuals. When a higher yield of plasmid or cosmid DNA was required, Midiprep DNA isolation kits were used according to manufacturer’s instructions. Obtained plasmid and cosmid DNA could be used for further restriction, PCR (polymerase chain reaction) or sequencing purposes.

Table 8: Commercial Mini- and Midiprep DNA isolation kits

Kit Name Sample size Company

QIAprep® Spin Miniprep kit 1-5 mL QIAGEN, Hilden, Germany QIAGEN® Plasmid Midi kit 100-500 mL QIAGEN, Hilden, Germany

peqGOLD Plasmid Miniprep kit 1-5 mL PEQLAB Biotechnology, Erlangen, Germany NucleSpin® Plasmid QuickPure 1-5 mL Macherey-Nagel, Dueren, Germany

Wizard® Plus SV Miniprep DNA

Purification System 1-5 mL Promega, Madison, Wisconsin, USA

4.1.2 Isolation of plasmid DNA by alkaline cell lysis

Alkaline cell lysis is used for isolation and separation of plasmid DNA from genomic DNA.

This method generates plasmid DNA in sufficient quality and quantity for further manipulation.

A preculture of E. coli harboring the desired plasmid was grown in 5 mL LB medium with appropriate antibiotic concentration at 37°C. 2-4 mL of this overnight culture were centrifuged for 3 min at 13000 rpm. Then the pellet was resuspended in 200 μL P1 buffer and cells were lysed by addition of 200 μL P2 buffer. After incubating at RT for maximum 5 min, the mixture was neutralized with 300 μL of P3 buffer and centrifuged for 10 min at 13000 rpm. The supernatant containing plasmid DNA was transferred into a new 1.5 mL Eppendorf Cup (hereafter E-Cup) and mixed with 500 μL chloroform/isoamyl alcohol (24:1, v/v). The two phases were mixed by shaking and for a new separation centrifuged for 5 min at 13000 rpm.

Then the upper layer was transferred into a new 1.5 mL E-Cup and plasmid DNA was precipitated with 500 μL cold isopropanol. The mixture was incubated on ice or at -20°C for 15 min and then centrifuged for 20 min at 13000 rpm, 4°C. The supernatant was pipetted off and the obtained pellet was washed two times each with 1 mL 70% ethanol by following centrifugation for 2 min at 13000 rpm, 4°C. The supernatant was discarded. The pellet was dried either at 37°C for 10-30 min or in a vacuum concentrator 5301 (Eppendorf, Hamburg, Germany) at 45°C for 3-5 min. The plasmid DNA was resuspended in 50 μL H₂O_bidest.

P1 buffer (sterile filtered) P2 buffer (sterile filtered)

Tris-HCl 1.21 g NaOH 4.0 g

EDTA 0.74 g SDS 5.0 g

RNase 100 µg/mL H2Obidest ad 500 mL H2Obidest ad 200 mL

pH 8.0

P3 buffer (sterile filtered) Kac 62.73 g H2Obidest ad 200 mL pH 5.5 (adjusted with acetic acid)

4.1.3 Isolation of plasmid DNA by cracking

Cracking is a very fast procedure resulting in unpurified plasmid DNA, thus this method is not suitable for further analysis such as sequencing. This procedure only provides information about the absence or presence of a plasmid.

As the cracking analysis requires little cell material, colonies had to be grown on LB plates containing appropriate antibiotics. Cell material was collected from the LB plate with a sterile pipette tip or a toothpick and resuspended in 25 μL 10 mM EDTA (pH 8.0). Then 25 μL freshly prepared cracking buffer were added and mixed well. The cell suspension was incubated for 5 min at 70°C and cooled on ice. Two μL of freshly prepared cracking dye were added and mixed. After incubation for 5 min on ice the cell suspension was centrifuged for 10 min at 13000 rpm, 4°C. Then, 20 μL of the supernatant were loaded on a 0.8% agarose gel (II.4.4) and electrophoresed at 100 V (Volt) for 1 h (hour).

Cracking buffer

2 N NaOH 100 μL

SDS (10%) 50 μL

Sucrose 0.2 g

H₂O_bidest 850 μL

Cracking dye

4 M KCl 150 µL

Bromophenol blue (0.4%) 50 µL

4.1.4 Isolation of cosmid DNA by the quick and dirty method

Quick and dirty isolation of cosmid DNA is based on the alkaline cell lysis method, which was already described in (II.4.1.2) with some minor modifications. Same buffers (P1, P2 and P3) were used for this cosmid DNA isolation.

A preculture of E. coli harboring the cosmid was grown in 5 mL LB medium with appropriate antibiotic concentration at 37°C. Five mL of this overnight culture were centrifuged for 30 sec at 9000 rpm. The pellet was resuspended in 100 μL P1 buffer and incubated at 37°C for 30 min up to 2 h. Cells were lysed by addition of 200 μL P2 buffer and incubated at room temperature (RT) for 1 min. After addition of 200 µL chloroform, the mixture was neutralized with 150 μL of P3 buffer, vortexed and centrifuged for 2 min at 13000 rpm. The supernatant containing cosmid DNA (approximately 400 µL) was transferred into a new 1.5 mL E-Cup and mixed with 1 mL ice cold 96% EtOH (corresponding to 2-3 volumes of supernatant). The phases were mixed by inverting several times and incubated at -20°C for 30 min. For a new separation, centrifugation was carried out for 20 min at 13000 rpm, 4°C. Then the supernatant was pipetted off and the obtained pellet was dried a few minutes at 37°C. The dry pellet was washed with 200 µL 70% ethanol by following centrifugation for 2 min at 13000 rpm, 4°C. The supernatant was discarded and the pellet was dried either at 37°C for 10-30 min or in a vacuum concentrator 5301 (Eppendorf, Hamburg, Germany) at 45°C for 3-5 min.

The cosmid DNA was resuspended in 20 μL H₂O_bidest.

4.1.5 Isolation of genomic DNA with AquaPure Genomic DNA kit

Purified genomic DNA from NGR234 was required for PCR amplification, sequencing and cloning. Therefore the AquaPure Genomic DNA kit (Bio-Rad Laboratories, Hercules, Canada) was applied according to manufacturer’s instructions.

4.1.6 Classical isolation of genomic DNA

A preculture of NGR234 was grown in 30 mL TY medium with rifampicin at 30°C for 2-3 days. Then 5-10 mL of this preculture were centrifuged for 30 sec at 9000 rpm. To remove excess medium and antibiotics, the pellet was resuspended in 1 mL NaCl. Then, cells were

harvested for 30 sec at 9000 rpm and resuspended in 250 µL TE-sucrose buffer (20%

sucrose dissolved in 1x TE buffer by heating in microwave). After adding 250 µL TE buffer containing lysozyme (10 mg/mL) and RNAase (1 mg/mL) the mixture was incubated for 1 h at 37°C. Proteinase K (1 mg/mL) as well as sarcosyl (overall volume of 5%) were added to the suspension and incubated for at least 1 h. After adding 250 µL phenol/chloroform (1:1) the suspension was mixed well until a white emulsion appeared. The emulsion was centrifuged for 20 min at 13000 rpm and the supernatant was carefully transferred into a new E-Cup. The phenol/chloroform step was repeated twice. Then, 250 µL of chloroform were added to the supernatant, mixed well and centrifuged for 2 min. The supernatant was transferred into a fresh E-Cup and participated with 2.5 volume of 96% EtOH and 0.3 M NaOAc. The E-Cup was inverted several times and incubated at -70°C for 10 min. After centrifugation for 20 min at 13000 rpm, the supernatant was decanted and mixed with 1 mL 70% EtOH and centrifuged again. This step was repeated once more. The DNA pellet was then dried, resuspended in 100 µL 1x TE buffer or H2Obidest and incubated for 1 h at 60°C or at RT overnight.