E. coli knockout

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Revision as of 09:12, 16 July 2010 (view source) Csbl (Talk | contribs) (→Lambda red recombinase method) ← Older edit		Revision as of 01:10, 21 July 2010 (view source) Igchoi (Talk | contribs) (→E. coli Gene Replacement) Newer edit →
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	===Lambda red recombinase method===		===Lambda red recombinase method===
-		+	(see [http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement this link] (openwetware::gene replacement) for the method comparison)
	==== Materials ====		==== Materials ====

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Revision as of 01:10, 21 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

(see this link (openwetware::gene replacement) for the method comparison)

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