Knockout1
From CSBLwiki
(Difference between revisions)
(→Materials) |
(→Materials) |
||
Line 3: | Line 3: | ||
==== Materials ==== | ==== Materials ==== | ||
- | *'''Plasmids''' - (see [http://compbio.korea.ac.kr/wiki/index.php/E._coli_knockout]) | + | *'''Plasmids''' - (see[http://compbio.korea.ac.kr/wiki/index.php/E._coli_knockout this link]) |
- | **pKD46, pKD13, pCP20 | + | **pKD46, pKD13, pCP20 |
*'''Target gene''' | *'''Target gene''' |
Revision as of 08:00, 15 October 2010
Contents |
Gene replacement
- target gene: yfaL
Materials
- Plasmids - (seethis link)
- pKD46, pKD13, pCP20
- Target gene
- yfaL
- yfaL
- Reagents
- L-arabinose
- Equipment
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℃.