Molecular Biological Methods

Cloning

Plasmid Preparation

Plasmid preparation is a method for isolating plasmids from cells. For plasmid preparation the illustra™ plasmidPrep MIni Spin Kit (GE Healthcare) was used. After cells are lysed, the lysate is applied to a mini column binding plasmid DNA to a silica membrane in the presence of chaotropic salts. After washing the DNA can be eluted with a low salt buffer. The kit was following the manufacturers instructions.

DNA-Purification

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