Knockout1

From CSBLwiki

Revision as of 02:52, 15 October 2010 (view source) Csbl (Talk | contribs) ← Older edit		Revision as of 02:53, 15 October 2010 (view source) Csbl (Talk | contribs) Newer edit →
Line 20:		Line 20:
	*'''Generation of cassette flanked by homologous arms'''		*'''Generation of cassette flanked by homologous arms'''
	**Design of primers		**Design of primers
		+	***
	**PCR condition		**PCR condition
-		+	***

---

Revision as of 02:53, 15 October 2010

Contents 1 Gene replacement 1.1 Materials 1.2 Procedure 1.3 Notes

Gene replacement

• target gene: yfaL

Materials

• Plasmids
  • pKD46
  • pKD13
  • pCP20
• 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 37℃.

Notes