Molecular biological methods 1 DNA analytical methods

UV spectrometry was used to measure DNA concentration at a wavelength of 260 nm, where one absorption unit corresponds to 50 ng/µl double stranded DNA in water. DNA-free water was used as a reference. Impurities present in the DNA can be determined by the 260/280 nm absorbance ratio, which should be approximately 1.8; deviations from this absorbance ratio indicate contaminations such as RNA or protein (Sambrook et al. 1989).

DNA samples were run on 1% (w/v) agarose in 1x TAE buffer supplemented with SYBR safe for DNA visualization. Before loading on the agarose gel, the DNA samples were mixed with 6x DNA-loading buffer. Agarose gel electrophoresis was carried out in 1x TAE at a constant voltage of 80 V. Cloned plasmid constructs were verified by restriction enzyme digest analysis and DNA-sequencing of the protein-coding regions by the Core Facility at the MPI of Biochemistry in Martinsried, Germany. The sequence chromatograms were visualized by the program Chromas (C. McCarthy, Griffith University, Australia) and the program

MultAlin (http://multalin.toulouse.inra.fr/multalin/multalin.html) was used for comparative sequence alignment analyses (Corpet 1988).

3.3.2

Competent E. coli cell preparation and transformation

3.3.2.1

Chemocompetent E. coli cells and chemical transformation

The RbCl method was used to prepare chemically competent E. coli cells (Hanahan 1983). 500 ml LB medium were inoculated with 10 ml of overnight culture of the respective

E. coli strain, and incubated at 37°C to an OD600 of 0.5. Cells were then kept for 15 min on ice

followed by centrifugation for 10 min at 1000 g and 4°C in sterilized centrifugation tubes. Supernatant was removed and pellet kept on ice. Cells were resuspended in 125 ml pre-cooled TFB I and incubated on ice for an additional 20 min. Subsequently, the cells were centrifuged again in sterile centrifugation tubes for 10 min at 1000 g and 4°C. Following supernatant decantation, the harvested cells were placed on ice and resuspended in 20 ml of pre-cooled TFB II. While keeping the cells on ice, 50 µl aliquots were added to pre-chilled tubes, frozen on dry ice, and stored at -80°C.

The prepared 50 µl chemocompetent cell aliquots were then used for heat shock transformation purposes. The 50 µl aliquots were thawed on ice and 50-100 ng of plasmid DNA or 10 µl of ligation reaction was added. The cells were incubated on ice for 30 min, followed by a 90 s heat shock at 42°C. After heat shock, the cells were once again incubated on ice for 5 min. 800 µl of LB was added to the cells and incubated for 1 hr at 37°C with gentle shaking. The cells were then plated on selective LB agar plates and incubated overnight at 37°C.

3.3.2.2

Electrocompetent E. coli cells and electroporation

Electrocompetent E. coli cells were made by inoculating 500 ml of LB medium with 2.5 ml overnight culture of the respective strain. Cells were grown to OD600 of 0.5 followed

by incubation on ice for 15 min. Next, cells were centrifuged in sterilized centrifuge tubes for 15 min at 4000 rpm (Beckmann centrifuge Avanti JLA 10.500 rotor) and 4°C. Supernatant was removed and harvested cells were resuspended in 500 ml cold sterile water followed by centrifugation as above. This washing step was repeated. After the second washing step, the cells were then resuspended in 40 ml pre-cooled, sterile 10% (v/v) glycerol. Centrifugation was carried out for 10 min at 3500 rpm (Beckmann centrifuge Avanti JLA 10.500) and 4°C. Harvested cells were then resuspended in 1 ml of pre-cooled, sterile 10% (v/v) glycerol and 50 µl was aliquoted to pre-chilled tubes, frozen on dry ice, and stored at -80°C.

Electroporation of the 50 µl electrocompetent E. coli cells was initiated by thawing the cells on ice and adding 50 ng of plasmid DNA to the cells. This was then transferred to a cooled 1 mm electroporation cuvette and pulsed (1250 V, 25 µF, 200 Ω). Directly after the pulse, the cells were taken up in 800 µl LB medium and incubated for 1 hr at 37°C with gentle shaking. The cells could then be plated on selective LB agar plates and incubated overnight at 37°C.

3.3.2.3

TSS transformation

This method was used for fast transformation purposes (Chung et al. 1989) and for transformations of more than one plasmid DNA in E. coli cells. The respective E. coli strain, potentially already transformed with one DNA plasmid, was plated on selective LB agar plates and grown overnight at 37°C; a single colony from the plate was used to inoculate 5 ml of LB medium containing respective antibiotic and accordingly incubated at 37°C to an OD600 of 0.4. An aliquot of 2 ml was centrifuged at 16,000 g for 1 min at RT; the supernatant

was decanted and the resulting pellet was resuspended in 100 µl TSS solution containing 0.5- 1 µg of plasmid DNA. After incubation on ice for 30 min, 0.5 ml of LB medium was added and the culture was incubated at 37°C with agitation. The cells could then be plated on selective LB agar plates and incubated overnight at 37°C.

3.3.3

Plasmid DNA and DNA fragment purification

E. coli DH5α was used for the amplification of plasmid DNA. The plasmid DNA was isolated and purified from overnight cultures grown in LB medium with appropriate antibiotics (Sambrook et al. 1989) by anion exchange chromatography using the Wizard Plus

SV Miniprep DNA Purification System or the PureYield Plasmid Midiprep System according to the manufacturer’s instructions.

Plasmid DNA or DNA fragments were purified after enzymatic reactions (e.g. after restriction digest, see section 3.3.6) or agarose gel electrophoresis (e.g. after PCR, see section 3.3.4) by anion exchange chromatography using the Wizard SV Gel and PCR Clean-Up System. Purification and isolation were performed according to the manufacturer’s instructions.

3.3.4

PCR (polymerase chain reaction)

Polymerase chain reaction was used to amplify DNA target sequences according to the standard protocol and cycling conditions indicated below in Table 3-1 and Table 3-2. When

necessary, appropriate modifications or adjustments were made to the standard protocol and/or PCR thermocycling conditions. Primer pairs used for PCR are listed in appendix 7.2.

Total volume 50 µl

Buffer 1x Pfu Polymerase buffer

dNTPs 0.25 mM

Primers 40 pmol each

Polymerase 3.75 U Pfu DNA Polymerase

Template DNA 100-200 ng plasmid or genomic DNA

Table 3-1 Standard PCR amplification protocol

Lid temperature of PCR thermocycler: 105°C

Step Purpose Temperature Duration Number of Cycles 1 Initial denaturation 95°C 300 s 1