E. coli knockout
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** Anneling temparature is tested at 55℃ at first. | ** Anneling temparature is tested at 55℃ at first. | ||
** Extension time is depend on the length of the target sequence. (1kb = about 1 min.) | ** Extension time is depend on the length of the target sequence. (1kb = about 1 min.) | ||
- | ** Detail conditions are | + | ** Detail conditions are shown below.[[Image:PCR condition.jpg|center|thumb|400px]] |
==== Notes ==== | ==== Notes ==== | ||
*Primers | *Primers |
Revision as of 07:48, 2 August 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
- Temperature sensitive replication origin
- 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
- Grow up pKD46, pKD13, and pCP20 in proper host strains
- Perform minipreps to extract plasmids
- Transformation of pKD46 to target strain
- Preparation of competent cells
- Using the protocol of TSS method
- Preparation of competent cells
- Generation of cassette flanked by homologous arms
- Design of primers
- Primers have overhangs which were homologous to surrounding region of target gene.
- Sequences from cassette region have about 60℃ Tm-value.
- Overview is attached. File:Overview
- Design of primers
- PCR condition
- Anneling temparature and extension time is depend on the target sequence.
- Anneling temparature is tested at 55℃ at first.
- Extension time is depend on the length of the target sequence. (1kb = about 1 min.)
- Detail conditions are shown below.
Notes
- Primers