Knockout1
From CSBLwiki
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*'''Electroporation''' | *'''Electroporation''' | ||
- | # Use 30㎕ competent cell and 300ng PCR product | + | # Use 30㎕ competent cell and 300ng PCR product. |
# Pulse 2.5kV, 200Ω, 25㎌. | # Pulse 2.5kV, 200Ω, 25㎌. | ||
# Add prewarmed 1㎖ LB broth immediately. | # Add prewarmed 1㎖ LB broth immediately. | ||
# Incubate 2 hour at 37℃. | # Incubate 2 hour at 37℃. | ||
- | # Spread one-tenth of the sample and incubate 37℃. | + | # Spread one-tenth of the sample and incubate at 37℃. |
==== Notes ==== | ==== Notes ==== |
Revision as of 07:54, 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 at 37℃.