E. coli knockout

From CSBLwiki

Revision as of 09:09, 16 July 2010 (view source) Csbl (Talk | contribs) (→Procedure) ← Older edit		Revision as of 09:09, 16 July 2010 (view source) Csbl (Talk | contribs) (→Notes) Newer edit →
Line 42:		Line 42:
	== Notes ==		== Notes ==
		+
		+	*Primers

---

Revision as of 09:09, 16 July 2010

Contents 1 E. coli Gene Replacement 1.1 Lambda red recombinase method 2 Materials 3 Procedure 4 Notes

E. coli Gene Replacement

Lambda red recombinase method

Materials

• Plasmids
  • pKD46
    • Coding lambda red recombinase
    • Ampiciline resistance
    • Temperature sensitive replication origin
  • pKD13
    • Kanamycin resistance gene template plasmid
  • pCP20
    • Flipase system
    • Ampiciline resistance
• Reagents
  • L-arabinose
    • Lambda red recombinase pBAD promoter inducer
• Equipment
  • Incubators (30°C and 37°C)
  • Electroporator

Procedure

• Preparation of plasmids

1. Grow up pKD46, pKD13, and pCP20 in host strains 
2. Perform minipreps to extract plasmids 

• Transformation of pKD46
  • Using the protocol of TSS method

• Generation of cassette flanked by homologous arms

1.
2.
3.
4.
5.

Notes

• Primers