E. coli knockout

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	*Generation of cassette flanked by homologous arms		*Generation of cassette flanked by homologous arms
-		+	**Design of primers
-	1.	+	***Primers have overhangs which were homologous to surrounding region of target gene.
-		+	***Sequences from cassette region have about 60℃ Tm-value.
-	2.	+	***Overview is attached.[[File:overview.jpg]]
-		+	#
-	3.	+	#
-		+
-	4.	+
-		+
-	5.	+
-		+
	==== Notes ====		==== Notes ====
	*Primers		*Primers

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Revision as of 07:24, 2 August 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 -sequence info.
    • 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
    • Temperature sensitive replication origin
• 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 proper host strains
2. Perform minipreps to extract plasmids

• Transformation of pKD46 to target strain
  • Preparation of competent cells
    • Using the protocol of TSS method

• Generation of cassette flanked by homologous arms
  • Design of primers
    • Primers have overhangs which were homologous to surrounding region of target gene.
    • Sequences from cassette region have about 60℃ Tm-value.
    • Overview is attached.

Notes

• Primers