Molecular Biological Methods

Cloning

Plasmid Preparation

Plasmid preparation is a method for isolating plasmids from bacterial cell cultures. In this work the illustra™ plasmidPrep MIni Spin Kit (GE Healthcare) was used. After the cells are lysed, the lysate is applied to a mini column binding plasmid DNA to a silica membrane in the presence of chaotropic salts. Following a washing step, the DNA is eluted with bidest. water. Unless stated otherwise the plasmid preparation was performed following the manufacturer’s manual.

DNA-Purification

Some molecular biological methods require a purification of DNA after amplification or modification. In this work, the illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare) was used. In the presence of chaotropic salts the nucleic acids are bound the glass fiber fleece in the Filter Tube while other substances are removed by the washing steps. Afterwards purified DNA fragments are be eluted with bidest. water. Unless stated otherwise the DNA purification was performed following the manufacturer’s manual.

Restriction Digest

Restriction endonucleases are used to cut double stranded DNA molecules at specific, usually palindromic base sequences. Unless stated otherwise the restriction digest was performed for 1 h at 37°C. To prevent religation of digested plasmids the DNA was dephosphorylated by addition of alkaline phosphatase and another incubation for 30 min at 37°C.

Ligation

Ligation in the context of molecular biology is the enzymatic joining of previously restricted nucleic acid fragments by synthesis of new bonds with simultaneous breakdown of ATP. A linearized plasmid functions as a vector for DNA inserts and all fragments are connected via ligation resulting in a new circular plasmid carrying the insert DNA. A typical reaction setup consists of a ligase, ligase buffer and the DNA fragments while the molar ratio of vector to insert should approximately be 1:3. The mixture was filled up with nuclease free water. Unless stated otherwise the reaction is incubated for 60 min at room temperature and is then used for transformation.

Gibson Assembly

Gibson assembly is a technique that allows fast isothermal assembly of multiple DNA fragments, regardless of fragment length. These fragments only need to have overlapping ends of 15-30 bp which can be created via PCR. Unless stated otherwise the Gibson Assembly was perfomed using the Gibson Assembly Cloning Kit (NEB) according to the protocol published by New England Biolabs. To design the primers for the respective PCRs a webtool was used (http://nebuilder.neb.com/).

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/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

Does the cells contain the correct insert/plasmid and does the insert have the expected length?'

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

Which are the optimal conditions for our primers to bind, the PCRs in generell?

Touchdown PCR

Avioding primer binding to non-specific sequences

SOE PCR

Method for side directed mutagenesis

QuikChange

Method for side directed mutagenesis

Conducted by Vera at the laboratories of the Schwaneberg Group. Supervision by Dr. rer. nat. Ljubica Vojcic.