E. coli knockout
From CSBLwiki
Contents |
E. coli Gene Replacement
Lambda red recombinase method
(see this link - openwetware::gene replacement - for the method comparison)
Materials
- Plasmids
- pKD46
- Coding lambda red recombinase
- Ampiciline resistance
- Temperature sensitive replication origin
- pKD13
- Kanamycin resistance gene template plasmid
- pCP20
- Flipase system
- Ampiciline resistance
- pKD46
- Reagents
- L-arabinose
- Lambda red recombinase pBAD promoter inducer
- L-arabinose
- Equipment
- Incubators (30°C and 37°C)
- Electroporator
Procedure
- Preparation of plasmids
1. Grow up pKD46, pKD13, and pCP20 in host strains 2. Perform minipreps to extract plasmids
- Transformation of pKD46
- Using the protocol of TSS method
- Generation of cassette flanked by homologous arms
1. 2. 3. 4. 5.
Notes
- Primers