E. coli knockout

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Revision as of 01:11, 21 July 2010 (view source) Igchoi (Talk | contribs) (→Lambda red recombinase method) ← Older edit		Revision as of 01:06, 30 July 2010 (view source) Igchoi (Talk | contribs) (→Materials) Newer edit →
Line 11:		Line 11:
	***Ampiciline resistance		***Ampiciline resistance
	***Temperature sensitive replication origin		***Temperature sensitive replication origin
-	**pKD13	+	**pKD13 - [http://www.ncbi.nlm.nih.gov/nuccore/AY048744.1 sequence info.]
-	***Kanamycin resistance gene template plasmid	+	***3.4kb, KanR flanking FRT sequence, R6K gamma replication origin
		+	***Kanamycin resistance gene template plasmid
	**pCP20		**pCP20
	***Flipase system		***Flipase system
Line 22:		Line 23:
	**Incubators (30°C and 37°C)		**Incubators (30°C and 37°C)
	**Electroporator		**Electroporator
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	==== Procedure ====		==== Procedure ====

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Revision as of 01:06, 30 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 - sequence info.
    • 3.4kb, KanR flanking FRT sequence, R6K gamma replication origin
    • 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