Knockout1

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		+	==Gene replacement==
		+	*target gene: yfaL
		+
		+	==== Materials ====
		+	*'''Plasmids'''
		+	**pKD46,  pKD13,  pCP20 - see [http://compbio.korea.ac.kr/wiki/index.php/E._coli_knockout this link]

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Revision as of 09:41, 15 October 2010

Contents 1 Gene replacement 1.1 Materials 1.2 Procedure 2 Gene replacement 2.1 Materials 2.2 Notes

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])

• Reagents
  • L-arabinose
    • Lambda red recombinase pBAD promoter inducer
• Equipment
  • Incubators (30°C and 37°C)
  • Electroporator

Procedure

• Preparation of plasmids
• Generation of cassette flanked by homologous arms
  • Design of primers
  • PCR condition

Gene replacement

• target gene: yfaL

Materials

• Plasmids
  • pKD46, pKD13, pCP20 - see this link

• Making competent cells

1. Pick the single colony containg pKD46.
2. Inoculate it to 5㎖ LB broth with ampicilline and L-arabinose.
   • L-arabinose final concentration is 1mM.
3. Incubate at 27℃, 180rpm until the culture OD reaches 0.6.
4. Centrifuge 3600rpm, for 5min at 4℃.
5. Wash with ice-cold 10% glycerol 3 times.
6. Concetrate 100-fold.

• Electroporation

1. Use 30㎕ competent cell and 300ng PCR product.
2. Pulse 2.5kV, 200Ω, 25㎌.
3. Add prewarmed 1㎖ LB broth immediately.
4. Incubate 2 hour at 37℃.
5. Spread one-tenth of the sample and incubate at 37℃.

Notes