VI. Material and Methods
1. Material
1.12 Disposables
Columns (Empty Reservoir 3 mL, 15 mL) Biotage
Cuvettes (Plastibrand 1.5 mL) Brand
96-Deepwell Plates TreffLab
384-Deepwell Plates ThermoScientific
Electroporation Cuvettes (Gene Pulser) BIORAD
Falcon (15 mL and 50 mL) Sarstedt, Peske
Gas Permeable Adhesive Seals ABgene
Glass Wool Serva
Gloves MaiMed medical, VWR
Petri Dishes Peske
Sealing Foils ABgene, 4titude
Sephadex G25 Columns GE Healthcare
Sterile Filter Paper Milipore
Syringes Brand, Henke Sass Wolf
Syringes Needles Braun, Mormject
Syringe Sterile Filters (0.2 µm, 0.45 µm) Peske
Tips Axygen
Tips Pipeting Robot Hamilton Robotics
TLC Plates (silica gel 60 F254) Merck
PCR Strips (0.2 mL) ThermoScientific
PCR-Tubes (1.5 and 2 mL) Sarstedt, Brand
PCR-Tubes (0. 2 mL) TreffLab
PCR-96-well Plates ABgene, Thermo Scientific
PCR 384-well Plates Roche
Sephadex A-25 DEAE Column Material Sigma
Whatman Chromatography Paper (3mm) GE Healthcare
2. Methods
2.1 Methods of Molecular Biology 2.1.1 PCR
Reaction mixtures contained 200 or 500 nM of the respective primers, 7.5-10 ng of the respective template/plasmid, 200 µM dNTPs, optional 3 % DMSO and 1 u Phusion DNA polymerase in 1x HF- or GC-buffer in a total volume of 50 µL. After initial denaturation for 30 or 60 s at 98 °C, 25-30 PCR cycles were performed with 10 s at 98 °C, 20-30 s at the respective hybridisation temperature, 130 s/kbp for elongation at 72 °C and one final elongation for 5-10 min at 72 °C.
2.1.2 Colony PCR
Colony PCR was employed to identify E. coli single colonies carrying a desired cloning product.
Thus, single colonies grown on LB agar plates over-night were picked and suspended in 10 µL water. The following PCR was performed with Taq DNA polymerase, kindly provided by Dr.
Ramon Kranaster. In short, 1 µL of bacterial suspension, 400 nM primers forward and reverse, 200 µM dNTPs and approximately 100 nM Taq DNA polymerase in 1x KlenTaq Reaction Buffer were mixed in a total volume of 20 µL. Amplification was performed with one initial denaturation step for 6 min at 95 °C, 30 PCR cycles with 30 s at 98 °C, 60 s at the respective hybridisation temperature, 2 min at 72 °C and alternatively with one final elongation step for 5 min at 72 °C. Product formation was analysed on a 0.8 % TBE agarose gel.
2.1.3 Site-directed Mutagenesis
Reactions were carried out according to the QuikChange Site-Directed Mutagenesis Kit protocol from Stratagene. In short, reaction mixtures contained 200 or 500 nM primers forward and reverse, containing the desired mutations, 25-50 ng of the respective template, 200 µM dNTPs, 3 % DMSO and 1 u Phusion DNA polymerase in 1x HF- or GC-buffer in a total volume of 50 µL. After initial denaturation for 60 s at 98 °C, 18 PCR cycles were performed with 10 s at 98 °C, 30 s at the respective hybridisation temperature, 3.5 min at 72 °C (30 s/kbp) and one final elongation for 10 min at 72 °C. Product formation was analysed on a 0.8 % TBE agarose gel. Next, methylated parental plasmid DNA was digested by adding 10-20 u DpnI and incubation for 3 h at 37 °C. Heat-inactivation of the enzyme was conducted at 80 °C for 20 min. Reactions were purified via a MinElute Reaction Cleanup-Kit (Qiagen) and subsequently transformed into E.coli BL21 (DE3) or XL10 Gold.
dephosphorylated and purified by preparative agarose gel electrophoresis using 0.8 % TAE agarose gels. Digestion of various amounts of PCR products followed the same procedure with the exception that the DNA was not dephosphorylated and solely purified after digestion via the MinElute Reaction Cleanup-Kit (Qiagen).
2.1.5 Dephosphorylation of double-stranded DNA
For 5’-dephosphorylation of digested plasmid DNA, 2 µL of Antarctic or Shrimp Alkaline Phosphatase and the respective buffer were added directly or alternatively after purification with the MinElute Reaction Cleanup-Kit (Qiagen) to the digested plasmid DNA (total volume 50 µL). After incubation for 2-3 h at 37 °C, the enzyme was heat-inactivated for 20 min at 80 °C. Afterwards, plasmid DNA was purified by preparative agarose gel electrophoresis using 0.8 % TAE agarose.
2.1.6 Ligation of DNA
For ligation of double-stranded DNA, 25 or 50 ng of pre-cut, dephosphorylated plasmid DNA were mixed with a 3- or 5- fold excess of DNA insert and 1 µL of the respective T4 DNA ligase in the supplied buffer. Ligation was either performed for 30 min at room temperature employing the Rapid Ligation Kit (Fermentas) or for 16 h at 16 °C using T4 DNA ligase from Roche. Insert DNA was omitted in the negative controls to test for religation of the vector. All reactions were purified via the MinElute Reaction Cleanup-Kit (Qiagen) preceding transformation into E. coli cells.
2.1.7 Isolation of Plasmid DNA
Plasmid DNA was isolated from 4 mL E. coli liquid cultures (chapter VI 2.3.4) grown over-night. Cells were harvested via centrifugation at 4 °C for 15 min and DNA isolated employing a plasmid Miniprep Kit. For isolation of higher amounts of DNA a plasmid Midiprep Kit from Qiagen was used according to the manufacturer’s instructions.
2.1.8 DNA Sequencing
Plasmid DNA was isolated and sent to GATC Biotech, Germany for sequencing. DNA concentrations varied in a range from 30-100 ng/µL. DNA sequences were analyzed using the ChromasLite software and SDSC Biology Workbench.
2.1.9 Analytical Agarose Gel Electrophoresis
DNA containing samples were mixed with 6x Agarose Loading Dye and separated on a 0.8 or 2.5 % agarose gel in 0.5x TBE buffer by applying 100-150 Volt. Agarose gels were selected according to the fragments size. After staining with ethidium bromide in 0.5x TBE buffer for 15-30 min and destaining in 0.5x TBE buffer for 15 min, the DNA was visualized by UV light using a Molecular Imager ChemiDoc XRS system (BIORAD).
2.1.10 Preparative Agarose Gel Electrophoresis
To purify DNA fragments, samples were mixed with 6x Agarose Loading Dye and separated on a 0.8 or 2.5 % agarose gel in 1x TAE buffer by applying 100 Volt. Agarose gels were selected according to the fragments size. After staining with ethidium bromide in 1x TAE buffer for 15-30 min and destaining in 1x TAE buffer for 15 min, the DNA was visualized by preparative UV light and excised with a scalpel. DNA was isolated from the agarose gel by employing a QIAquick Gel Extraction Kit (Qiagen).
2.1.11 Analytical Denaturing PAGE
Analytical denaturing PAGE was employed to separate and analyze DNA oligonucleotides.
First, a 12 % PAGE-gel was prepared by mixing 48 mL 1x Denaturing PAGE Gel Solution I, 42 mL 1x Denaturing PAGE Gel Solution II, and 10 mL 1x Denaturing PAGE Gel Solution III with 800 μL 10 % APS and 40 μL TEMED. The solution was then applied to the gel chamber.
Final gel thickness was 0.4 mm. DNA samples were supplied with 1x Denaturing PAGE Loading Dye (stop solution) in a 2.25 fold excess and 1.5 µL (per well) of the sample was loaded onto the gel. DNA fragments were then separated in 1x TBE buffer by applying 100 W and 3000 V at up to 50 °C. Subsequently, the gel was transferred to whatman paper, dried in vacuo at 80 °C for at least 45 min and exposed to a phosphor imager screen over-night.
Readout was facilitated using the Molecular Imager FX (BIORAD).
2.1.12 Preparative Denaturing PAGE
Preparative denaturing PAGE was employed to purify DNA oligonucleotides. PAGE-gels were prepared (8-12 %) according to the DNA fragment’s size. First, respective amounts of 1x
filled with silanised glass-fibres wool. Oligonucleotides were then ethanol precipitated, dissolved in ddH2O and concentrations determined by absorption measurements at 260 nm.