Knockout1
From CSBLwiki
(Difference between revisions)
(→Gene replacement) |
|||
Line 17: | Line 17: | ||
==== Procedure ==== | ==== Procedure ==== | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | *'''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℃. | ||
Revision as of 02:51, 15 October 2010
Contents |
Gene replacement
- target gene: yfaL
Materials
- Plasmids
- pKD46
- pKD13
- pCP20
- pKD46
- Reagents
- L-arabinose
- Equipment
Procedure
- 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℃.