Knockout1
From CSBLwiki
(Difference between revisions)
(→Materials) |
|||
Line 5: | Line 5: | ||
*'''Plasmids''' | *'''Plasmids''' | ||
**pKD46 | **pKD46 | ||
- | |||
**pKD13 | **pKD13 | ||
- | |||
**pCP20 | **pCP20 | ||
+ | |||
+ | *'''Target gene''' | ||
+ | **yfaL | ||
*** | *** | ||
+ | |||
*'''Reagents''' | *'''Reagents''' | ||
**L-arabinose | **L-arabinose | ||
+ | |||
*'''Equipment''' | *'''Equipment''' | ||
** | ** |
Revision as of 07:39, 15 October 2010
Contents |
Gene replacement
- target gene: yfaL
Materials
- Plasmids
- 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 37℃.