E. coli knockout

From CSBLwiki

Revision as of 04:07, 30 July 2010 (view source) Igchoi (Talk | contribs) (→Materials) ← Older edit		Revision as of 07:01, 2 August 2010 (view source) Csbl (Talk | contribs) Newer edit →
Line 16:		Line 16:
	**pCP20		**pCP20
	***Flipase system		***Flipase system
-	***Ampiciline resistance	+	***Ampiciline resistance
		+	***Temperature sensitive replication origin
	*'''Reagents'''		*'''Reagents'''
	**L-arabinose		**L-arabinose
Line 27:		Line 28:
	*Preparation of plasmids		*Preparation of plasmids
-	1. Grow up pKD46, pKD13, and pCP20 in host strains
-	2. Perform minipreps to extract plasmids
-	*Transformation of pKD46	+	# Grow up pKD46, pKD13, and pCP20 in proper host strains
-	**Using the protocol of TSS method	+	# 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		*Generation of cassette flanked by homologous arms
-	1.	+
-	2.	+	1.
-	3.	+
-	4.	+	2.
-	5.	+
		+	3.
		+
		+	4.
		+
		+	5.

---

Revision as of 07:01, 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

1.

2.

3.

4.

5.

Notes

• Primers