Molecular biological methods

Cloning

Restriction Digest

Ligation

Gibson Assembly

The Gibson Assembly was conducted according to the protocol published by New England Biolabs.

1. Set up the reaction according to the table below on ice (2-3 fragment assembly).
2. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
3. Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.

Transformation

Heat Shock

1. thaw cells on ice
2. add 1 µL of plasmid DNA
3. incubate on ice for 30 min
4. heat shock at 42 °C for 60 s
5. incubate on ice for 5 min
6. add 200 µL of SOC media
7. incubate at 37 °C for 2 h
8. plate 20  and 200 µL on plates supplemented with the appropiate antibiotic

Electroporation

1. add 1 μL plasmid to electrocompetent cells
2. put DNA/ cell suspension in electroporation cuvette
3. wipe dry the electroporator
4. use a small plastic pipette to place the cells
5. pulse: 2.5 kV, 200-400 Ω, 25 μF (for E.coli)
6. immediatly add 1 mL LB and incubate for 2 h at 37 °C
7. plate 50 μL on selective medium plate
8. centrifuge the rest (3000 g, 20 min), discard supernatant, re-suspend the pellet in 50 μL LB and plate it on selective medium plate

PCR

We have used several different types of PCR throughout our project:

• colony PCR
• check PCR
• gradient PCR
• SOE PCR
• touchdown PCR
• QuikChange(Ligation-During-Amplification)

The scope of appplication as well as the conduct are described below.

Colony PCR /Check PCR

With GoTaq Mast Mix

• 12.5 µl GoTaq Master Mix
• 1 µl primer_F
• 1 µl primer_R
• pick colony with tip and suspend in PCR tube
• 9.5 µl ddH₂O

gradient PCR

SOE PCR

touchdown PCR

QuikChange

Analytical methods

Agarose gel electrophoresis

Separation of DNA or RNA

1. take 5µl of the PCR product
2. mix with 1µl loading dye
3. apply onto agarose gel together with a marker
4. run at 120°C for 40 minutes for a full gel

SDS-PAGE

Cell preparation

• lysis cell pellet in lysis buffer
• centrifuge for 15 min at 13.000 rpm
• mix the supernatant with 2x lammli buffer with β-mercaptoethanol
• denatured for 5 min at 95 °C
• sample to the gel

For some SDS-PAGEs, we used BioRad ready made gels.

The recipe of the self-made SDS is as follows:

1.5x Buffer

• 1.5 M Tris-Cl pH = 8.8
• in 1 L is 40 ml 10 % SDS

Gels

Run gel

• apply the prepared samples together with a protein marker on the gel
• run the gel for 10 min at 60 V and after that for ca. 60 min at 120 V

Bradford assay

Determination of protein concentration

Measurement of fluorescence

The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.

• volume of sample in each well: 100µl
• measure GFP fluorescence at an excitation wavelength of 496 nm and an emission wavelength at 516 nm

Measurement of optical density

Depending on the number of samples, two different devices were used for measurement of optical density, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx (BioTek) microplate reader.