Knockout1
From CSBLwiki
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==== Notes ==== | ==== Notes ==== | ||
+ | *'''Primers''' | ||
+ | ** For pKD 13 kanamycin cassette | ||
+ | *** Foward: ATGCGGATTATCTTTCTACGCAAGGAGTATTTATCTTTACaATTCCGGGGATCCGTCGACC | ||
+ | *** Reverse: gcgcccctgccagcacgtagtgtgtaggctggagctgcttcggccggttagcgccatcggct | ||
+ | ** For confirmation of Knockout | ||
+ | *** Foward: GTTTCATGCAGACTACCTGTAGTACG | ||
+ | *** Reverse: ctgcctcgtcctgcagttcattca | ||
+ | |||
+ | *'''Sequences''' | ||
+ | **pKD13 Kanamycin cassette with FRT |
Revision as of 08:36, 21 October 2010
Contents |
Gene replacement
- target gene: yfaL
Materials
- Plasmids
- pKD46, pKD13, pCP20 - see this link
- Target gene
- yfaL
- an AmpR and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis (Datsenko and Wanner paper [1])
- yfaL
- Reagents
- L-arabinose
- Lambda red recombinase pBAD promoter inducer
- L-arabinose
- Equipment
- Incubators (30°C and 37°C)
- Electroporator
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℃.
Notes
- Primers
- For pKD 13 kanamycin cassette
- Foward: ATGCGGATTATCTTTCTACGCAAGGAGTATTTATCTTTACaATTCCGGGGATCCGTCGACC
- Reverse: gcgcccctgccagcacgtagtgtgtaggctggagctgcttcggccggttagcgccatcggct
- For confirmation of Knockout
- Foward: GTTTCATGCAGACTACCTGTAGTACG
- Reverse: ctgcctcgtcctgcagttcattca
- For pKD 13 kanamycin cassette
- Sequences
- pKD13 Kanamycin cassette with FRT