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E. coli Gene Replacement
Lambda red recombinase method
Materials
- Plasmids
- pKD46
- Coding lambda red recombinase
- Ampiciline resistance
- Temperature sensitive replication origin
- pKD13
- Kanamycin resistance gene template plasmid
- pCP20
- Flipase system
- Ampiciline resistance
- Reagents
- L-arabinose
- Lambda red recombinase pBAD promoter inducer
- Equipment
- Incubators (30°C and 37°C)
- Electroporator
Procedure
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
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Notes