NUCLEIC ACID ANALYSIS 27

2.2.1.1 DNA ANALYSIS

2.2.1.1.1 ISOLATION OF TOTAL DNA

Oenothera produces high amounts of tannins and mucilage (Zinsmeister et al. 1965, 1970), which provide the major obstacle to DNA extraction from Oenothera. It was therefore necessary to avoid the co-precipitation of polysaccharides with DNA. The presence of these polysaccharides prevents complete redissolution of DNA. Furthermore, polysaccharides prevent enzyme activities, thus inhibiting processes

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such as cutting with restriction enzymes, PCR or in vitro labeling (Barnwell et al.

1998). It has been proposed to use hydrolytic enzymes (Rether et al. 1993) or ion- exchange resins (Guillemaut and Maréchal-Drouard 1992; Maréchal-Drouard and Guillemaut 1995) to remove polysaccharides from nucleic acid solutions.

The genomic DNA was extracted from plants growing either in a greenhouse or on a field. DNeasy Plant Mini Kit in combination with a 96-well ball mill (Qiagen, Hilden, Germany) allows an efficient large-scale DNA isolation procedure for Oenothera

tissue. The standard protocol was slightly modified: 50 - 100 mg fresh leaf material was disrupted in 2 ml reaction tubes in the presence of 400 μl of buffer AP1, 4 μl RNase A (100 mg/ml), 4 μl 10% PVP (polyvinyl pyrrolidone) and 0.4 µl 1 M sodium 1- ascorbic acid, using a Mixer Mill MM 300 (Qiagen, Hilden, Germany) (2 times 2 min at 30 Hz). The samples were subsequently incubated for 10 min at 65°C to lyse the cells. During incubation, the samples were mixed 2 - 3 times by inverting the tube. The homogenized leaf material was centrifuged (1 - 2 min at 4,000 x g). 130 µl of buffer AP2 was added to the lysate, mixed, and incubated for 5 min on ice to precipitate detergent, proteins and polysaccharides, followed by another centrifugation step (5 min at 20,000 x g). This step is crucial to DNA isolation of leaf material from Oenothera plants. Oenothera generates highly viscous lysates and large amounts of precipitates during this step. This can result in shearing DNA in the following step. Satisfactory results were obtained, because the majority of precipitates were removed by this centrifugation step. The supernatant was pipetted into the QIAshredder Mini spin column that was placed in a 2 ml collection tube, and centrifuged for 2 min at 20,000 x g. The column removes most of the remaining precipitate and cell debris but still a rest passed through and formed a pellet in the collection tube. The flow-through fraction was carefully placed in a new 2 ml collection tube without disturbing the cell-debris pellet. 1.5 volumes of buffer AP3/E was added to the lysate and mixed immediately by pipetting. The mixture was transferred into the DNeasy Mini spin column (a silica-based membrane) that was placed in a 2 ml collection tube and centrifuged for 1 min at ≥ 6,000 x g. The flow- through was discarded and the column was placed into a new 2 ml collection tube. 500 µl of buffer AW was added and centrifuged for 1 min ≥ 6,000 x g. This step was repeated twice, followed by another centrifugation step (2 min at 20,000 x g) to dry

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the membrane and remove the complete ethanol that is the essential element in buffer AW. The column was transferred to a 1.5 ml tube and 30 - 50 µl of buffer AE were directly pipetted onto the DNeasy membrane. After an incubation (5 min at room temperature), the column was centrifuged (1 min 6,000 x g) to elute DNA. All columns and buffers used were components from DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). DNA concentration and quality was estimated by spectrophotometry and ethidium bromide stained agarose gel electrophoresis.

2.2.1.1.2 PCR PRODUCT PURIFICATION

PCR products were purified using either “QiaQuick Purification Kit” according to manufacturer´s manual or by standard precipitation via Na-acetate (pH 5.2) and 100% EtOH.

2.2.1.1.3 PLASMID TRANSFORMATION

50 - 100 µl MAX Efficiency® DH5α Competent Cells (Invitrogen, Karlsruhe, Germany) (Bethesda Research Laboratories 1986) were carefully unfrozen on ice. The required volume of DNA (vector) from the ligation reaction was added and the suspension gently mixed with a pipette. Subsequently to an incubation period for 30 min on ice, cells were exposed to a heat shock (40 - 50 sec at exactly 42°C in a water bath) followed by an incubation for 5 min on ice again. 0.2 – 0.4 ml of SOC medium (Sambroock and Russell 1989) were added and the suspension was shaken at 225 rpm at 37°C for 1 h. 50 µl of the cells were plated on LB medium (Bertani 1951) plates with ampicillin and incubated for 14 – 18 h at 37°C

2.2.1.1.4 PLASMID ISOLATION

Plasmids were isolated using the “Qiagen Plasmid Mini Kit” according to the manufacturer´s protocol.

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2.2.1.2 AUTOMATED SEQUENCING ON THE ABI PRISM 377 DNA SEQUENCER

Table 2-1: Pipetting scheme of sequencing gels

H2Obidest 22.8 ml Urea 18 g 10x TBE 6 ml Accugel 29:1 40% 5.33 ml APS (15%) 300 µl TEMED 20 µl

The dideoxy sequencing method (Sanger et al. 1977) was applied for automated sequencing using the ABI PRISM 377 DNA sequencer. The DNA was labeled with “Thermo SequenaseTM Dyenamic Direct sequencing kits”. The following conditions were used: 1 cycle of 95°C denaturation for 2 min; 99 cycles at 95°C (10 sec), 49 - 55°C (5 sec), and 1 cycle 60°C (4 min). The samples were cleaned up by ethanol precipitation to remove unincorporated fluorescent. The pellet was resuspended in 4 µl loading buffer (80% formamide, 10 mg/ml Dextran Blue and 5 mM EDTA), followed by a denaturation step at 80°C for 2 min. After denaturation the sample can be stored at 4°C. The sequencing gel was prepared as described in Table 2-1. The sequence run was performed using 48 cm glass plates, 0.2 mm spacers and a 36 well comb. Settings for electrophoresis conditions were 2.5 kV, 45°C and 11 h using 1x TBE electrophoresis buffer (89 mM Tris, 89 mM boric acid and 2 mM EDTA, pH 8.3).