DNA techniques

2.2.2.1 Purification of plasmid DNA

Plasmid DNA was purified with the Pharmacia GFX Micro Plasmid Kit in small scale and the Qiagen Plasmid Maxi Kit or the Nucleobond Kit in large scale according to the manufacturer’s instructions.

2.2.2.2 Determination of DNA concentration

The concentration and purity of the purified DNA was determined by measuring the UV absorbance at 260 and 280 nm. The DNA concentration was calculated with the OD260nm

(1 OD_260nm = 50 µg/ml dsDNA or 33 µg/ml ssDNA). The purity was estimated with the OD260nm/OD280nm ratio, with a ratio of ca. 1.8 indicating a low degree of protein contamination. 2.2.2.3 Restriction endonuclease digestion

Restriction endonuclease reactions were performed according to the manufacturer’s recommendations. In general, 1 µg DNA was digested for 1 h at 37°C with 1-3 U enzyme. Efficacy of the cleavage reaction was controlled by agarose gel electrophoresis.

2.2.2.4 5’-Dephosphorylation reaction

5’-dephosphorylation reaction of plasmid vector DNA after restriction endonuclease cleavage was performed with the shrimp alkaline phosphatase (SAP). 2 U SAP were added to about 2 µg restriction enzyme digested plasmid DNA. After 30 min incubation at 37 °C the phosphatase was inactivated by heating to 65°C for 15 min and the DNA was isolated by phenol/chloroform extraction and ethanol precipitation (see below).

2.2.2.5 Polymerase chain reaction (PCR)

Polymerase Chain Reaction (PCR) was performed with the Pwo DNA polymerase from Pyrococcus woesei with a 3’→5’ exonuclease proofreading activity.

The reaction mixture contained:

10 µl 10x PCR Puffer (100 mM Tris-HCl pH 8.85, 250 mM KCl, 50 mM (NH4)2SO4, 20 mM MgSO₄) 2 µl 10 mM dNTPs (200 µM each) 2 µl 10 µM sense primer (200 nM) 2 µl 10 µM antisense primer (200 nM) 0.5 µl Pwo (2.5U) 83.5 µl H2O + 0.1 µg template DNA

The following cycles were performed:

a) Amplification of the region encoding the N-terminal part of E3/49K for the cloning of the His-Tag-fusion construct: 1. 94°C 5 min 2. 94°C 1 min 3. 55° C 1 min 25x 4. 72°C 2 min 5. 72°C 10 min

b) Amplification of the region encoding the C-terminal part of E3/49K and site-directed mutagenesis:

1. 94°C 5 min 2. 94 °C 1 min

3. 62°C 2 min 12x with a decrease of 1°C per cycle down to 50°C (touchdown), then 15x 4. 72°C 2 min

5. 72°C min 10 min

2.2.2.6 Isolation of DNA fragments

DNA fragments were separated by agarose gel electrophoresis, stained with ethidium bromide and detected with UV light (366 nm). The gel slice containing the DNA fragments was cut out and the DNA was isolated using the QIAex II Agarose Gel Extraction Kit according to the manufacturer’s instructions. Alternatively, DNA fragments from PCR reactions were purified using the QIAquick PCR Purification Kit according to the manufacturer’s instructions.

2.2.2.7 Phenol/chloroform extraction and ethanol precipitation

Proteins were removed from DNA preparations by extracting twice with 1x volume phenol/chloroform and once with 1x volume chloroform. After vigorous vortexing for 10 s the solution was centrifuged at 14000 rpm (microcentrifuge) for 1 min and the upper DNA containing phase was recovered. Then 0.1x volume 3 M NaAc pH 5.2 and 3x volume 100% EtOH (cold) were added, and incubation at –20°C was performed for 1-12 h. The precipitated DNA was centrifuged down at 14000 rpm for 30 min (4°C). Then the pellet was washed once with 70% EtOH (cold). After another centrifugation step (14000 rpm, 15 min, 4°C, microcentrifuge) the EtOH was carefully removed, the pellet air-dried at RT and finally resuspended in H2O or 10 mM Tris pH 8.0.

2.2.2.8 Ligation

For ligation about 100-200 ng vector DNA was used with a molar ration of vector/insert of about 1:1. The reaction was performed in a total volume of 20 µl 1x reaction buffer (MBI Fermentas) with 2 U T4 DNA Ligase (MBI Fermentas).

In the first step vector and insert were mixed in reaction buffer and incubated 5 min at 45°C and put on ice. Then the ligase was added. After incubation at 1 h 22°C the ligase was inactivated by heating 10 min at 65 °C.

2.2.2.9 DNA sequencing 2.2.2.9.1 PCR

DNA sequencing was performed using the BigDye RR Terminator Amplitaq FS Kit. The reaction mixture contained 8 µl Premix, 1 µg dsDNA template and 10 pmol primer in a total

volume of 20 µl.

Then the PCR was performed: 96°C 10 s

50°C 5 s 25x 60°C 4 min

The product was precipitated by adding 30 µl H₂O, 5 µl 3 M NaAc pH 5.2 and 135 µl EtOH (RT). After 15 min centrifugation at 15000 rpm the pellet was washed with 280 µl 70% EtOH (RT). Then it was centrifuged for 10 min at 14000 rpm (microcentrifuge), the EtOH was removed and the pellet air-dried.

2.2.2.9.2 Polyacrylamide gel electrophoresis

A 5% polyacrylamide gel (8 M urea, 200 x 560 x 0.3 mm) was prepared as follows: 30 g urea

10 ml 30% acrylamide/bisacrylamide (29:1) 6 ml 10x TBE buffer

22 ml H2O

10x TBE buffer (1l): 108 g Tris base 55 g Boric acid 7.4 g EDTA

The solution was incubated at 37°C until the urea was dissolved. Then the solution was filtered with a 0.2 µm filter and degassed for 10 min. 20 µl TEMED and 350 µl 10% APS solution were added and the gel was poured and polymerized horizontally for 4 h.

The precipitated DNA from the PCR reaction was resuspended in 2 µl Blue Dextran/EDTA (50 mg/ml Blue Dextran, 25 mM EDTA pH 8) and 8 µl formamide and heated for 10 min at 95°C. Then the samples were put on ice for 5 min. After 1 min centrifugation at 14000 rpm (microcentrifuge) 4 µl of the solution was loaded on the gel. The gel run was performed with 1x TBE buffer at 37 Watt for 18 h (prerun 30 min). Sequencing data were analyzed using the ABI PRISM software.

2.2.2.10 Agarose gel electrophoresis

Analysis of DNA fragments and plasmids was performed by agarose gel electrophoresis in 1x TAE. In general, agarose concentration was between 1 and 2 % in 1x TAE. The agarose was solubilized by heating in a microwave oven. Ethidium bromide was added to a final concentration of 0.1 µg/ml (1 µl stock to 100 ml) just before pouring the gel. Probes were mixed with 0.17x volume loading buffer. Gels (6.5 x 9.5 cm) were run horizontally at 80-120 V. DNA was detected with UV light, λ=254 nm or λ=366 nm to cut out specific fragments.

Loading buffer (6x in water): 0.25% bromophenol blue 0.25% xylene cyanol FF 15% Ficoll (type 400) 20x TAE: 800 mM Tris 400 mM NaAc 40 mM EDTA

adjusted to pH 7.8 with acetic acid Ethidium bromide (stock): 10 mg/ml