Molecular biological methods
Cloning
Restriction Digest
Ligation
Gibson Assembly
The Gibson Assembly was conducted according to the protocol published by New England Biolabs.
- Set up the reaction according to the table below on ice (2-3 fragment assembly).
- 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.
- Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
Total Amount of Fragments | 0.02-0.5 pmols
|
Gibson Assembly Master Mix (2X) | 10 µl
|
Deionized H2O | 10-X µl
|
Total Volume | 20 µl
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Transformation
Heat Shock
- thaw cells on ice
- add 1 µL of plasmid DNA
- incubate on ice for 30 min
- heat shock at 42 °C for 60 s
- incubate on ice for 5 min
- add 200 µL of SOC media
- incubate at 37 °C for 2 h
- plate 20 and 200 µL on plates supplemented with the appropiate antibiotic
Electroporation
- add 1 μL plasmid to electrocompetent cells
- put DNA/ cell suspension in electroporation cuvette
- wipe dry the electroporator
- use a small plastic pipette to place the cells
- pulse: 2.5 kV, 200-400 Ω, 25 μF (for E.coli)
- immediatly add 1 mL LB and incubate for 2 h at 37 °C
- plate 50 μL on selective medium plate
- 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
parameter | duration | temp [°C] |
|
denature | 5:00 | 95 |
|
anneal | 00:30 | 56 | 30 cycles
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elongate | 01:00 per kb | 72
|
denature | 00:30 | 95
|
elongate | 05:00 | 72 |
|
store | forever | 8
|
gradient PCR
SOE PCR
touchdown PCR
QuikChange
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