E. coli knockout

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	===Lambda red recombinase method===		===Lambda red recombinase method===
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	==== Materials ====		==== Materials ====
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	**Incubators (30°C and 37°C)		**Incubators (30°C and 37°C)
	**Electroporator		**Electroporator
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	==== Procedure ====		==== Procedure ====
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	==== Notes ====		==== Notes ====
	*Primers		*Primers

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Revision as of 09:11, 16 July 2010

Contents 1 E. coli Gene Replacement 1.1 Lambda red recombinase method 1.1.1 Materials 1.1.2 Procedure 1.1.3 Notes

E. coli Gene Replacement

Lambda red recombinase method

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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

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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.

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Notes

• Primers