Knockout1
From CSBLwiki
(Difference between revisions)
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==== Procedure ==== | ==== Procedure ==== | ||
+ | *'''Preparation of plasmids''' | ||
+ | *'''Generation of cassette flanked by homologous arms''' | ||
+ | **Design of primers | ||
+ | **PCR condition | ||
Revision as of 02:52, 15 October 2010
Contents |
Gene replacement
- target gene: yfaL
Materials
- Plasmids
- pKD46
- pKD13
- pCP20
- pKD46
- Reagents
- L-arabinose
- Equipment
Procedure
- Preparation of plasmids
- Generation of cassette flanked by homologous arms
- Design of primers
- PCR condition
- 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 37℃.