Knockout1
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==== Materials ==== | ==== Materials ==== | ||
| - | *'''Plasmids''' - | + | *'''Plasmids''' |
| - | + | **pKD46, pKD13, pCP20 - see [http://compbio.korea.ac.kr/wiki/index.php/E._coli_knockout this link] | |
*'''Target gene''' | *'''Target gene''' | ||
**yfaL | **yfaL | ||
| - | *** | + | ***an AmpR and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis (Datsenko and Wanner paper [http://www.pnas.org/content/97/12/6640.full]) |
*'''Reagents''' | *'''Reagents''' | ||
**L-arabinose | **L-arabinose | ||
| - | + | ***Lambda red recombinase pBAD promoter inducer | |
*'''Equipment''' | *'''Equipment''' | ||
| - | ** | + | **Incubators (30°C and 37°C) |
| + | **Electroporator | ||
==== Procedure ==== | ==== Procedure ==== | ||
Revision as of 08:07, 15 October 2010
Contents |
Gene replacement
- target gene: yfaL
Materials
- Plasmids
- pKD46, pKD13, pCP20 - see this link
- Target gene
- yfaL
- an AmpR and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis (Datsenko and Wanner paper [1])
- yfaL
- Reagents
- L-arabinose
- Lambda red recombinase pBAD promoter inducer
- L-arabinose
- Equipment
- Incubators (30°C and 37°C)
- Electroporator
Procedure
- Preparation of plasmids
-
- Generation of cassette flanked by homologous arms
- Design of primers
- PCR condition
- Design of primers
- Making competent cells
- Pick the single colony containg pKD46.
- Inoculate it to 5㎖ LB broth with ampicilline and L-arabinose.
- L-arabinose final concentration is 1mM.
- Incubate at 27℃, 180rpm until the culture OD reaches 0.6.
- Centrifuge 3600rpm, for 5min at 4℃.
- Wash with ice-cold 10% glycerol 3 times.
- Concetrate 100-fold.
- Electroporation
- Use 30㎕ competent cell and 300ng PCR product.
- Pulse 2.5kV, 200Ω, 25㎌.
- Add prewarmed 1㎖ LB broth immediately.
- Incubate 2 hour at 37℃.
- Spread one-tenth of the sample and incubate at 37℃.
