E. coli knockout
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*'''Plasmids''' | *'''Plasmids''' | ||
- | **pKD46 | + | **pKD46 -[http://www.ncbi.nlm.nih.gov/nuccore/AY048746.1 sequence info.] |
***Coding lambda red recombinase | ***Coding lambda red recombinase | ||
***Ampiciline resistance | ***Ampiciline resistance |
Revision as of 04:07, 30 July 2010
Contents |
E. coli Gene Replacement
Lambda red recombinase method
(see this link - openwetware::gene replacement - for the method comparison)
Materials
- Plasmids
- pKD46 -sequence info.
- Coding lambda red recombinase
- Ampiciline resistance
- Temperature sensitive replication origin
- pKD13 - sequence info.
- 3.4kb, KanR flanking FRT sequence, R6K gamma replication origin
- Kanamycin resistance gene template plasmid
- pCP20
- Flipase system
- Ampiciline resistance
- pKD46 -sequence info.
- 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