Knockout1

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Revision as of 07:54, 15 October 2010 (view source) Csbl (Talk | contribs) (→Procedure) ← Older edit		Revision as of 07:58, 15 October 2010 (view source) Csbl (Talk | contribs) (→Materials) Newer edit →
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	==== Materials ====		==== Materials ====
-	*'''Plasmids'''	+	*'''Plasmids''' - (see [http://compbio.korea.ac.kr/wiki/index.php/E._coli_knockout])
-	**pKD46	+	**pKD46, pKD13, pCP20
-	**pKD13	+
-	**pCP20	+
	*'''Target gene'''		*'''Target gene'''

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Revision as of 07:58, 15 October 2010

Contents 1 Gene replacement 1.1 Materials 1.2 Procedure 1.3 Notes

Gene replacement

• target gene: yfaL

Materials

• Plasmids - (see [1])
  • pKD46, pKD13, pCP20

• Target gene
  • yfaL

• Reagents
  • L-arabinose

• Equipment

Procedure

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

• 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