E. coli knockout

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	**Treatment of PCR products		**Treatment of PCR products
	***Method 1.		***Method 1.
		+	****Takes more time and product concentration is lower than method 2. But template vectors remain less.
		+
	# PCR products were gel-purified.		# PCR products were gel-purified.
	# Digested with DpnI for 2~3 hours.		# Digested with DpnI for 2~3 hours.
	# Repurified and suspended in elution buffer (10mM Tris, pH8.0)		# Repurified and suspended in elution buffer (10mM Tris, pH8.0)
-	****Takes more time and product concentration is lower than method 2. But template vectors remain less.	+
	***Method 2.		***Method 2.
		+	****Takes less time and get high yield of products. Templates can be remained.(But it's not affect critically to transformation.)
		+
	# Add 1㎕ of DpnI to not purified PCR products.		# Add 1㎕ of DpnI to not purified PCR products.
	# Incubate at 37℃ about 2~3 hours.		# Incubate at 37℃ about 2~3 hours.
	# This samples were gel-purified and suspended in elution buffer.		# This samples were gel-purified and suspended in elution buffer.
-	****Takes less time and get high yield of products. Templates can be remained.(But it's not affect critically to transformation.)
	==== Notes ====		==== Notes ====
	*Primers		*Primers

---

Revision as of 08:04, 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. File:Overview
  • PCR condition
    • Anneling temparature and extension time is depend on the target sequence.
    • Anneling temparature is tested at 55℃ at first.
    • Extension time is depend on the length of the target sequence. (1kb = about 1 min.)
    • Detail conditions are shown below.
      

      
  • Treatment of PCR products
    • Method 1.
      • Takes more time and product concentration is lower than method 2. But template vectors remain less.

1. PCR products were gel-purified.
2. Digested with DpnI for 2~3 hours.
3. Repurified and suspended in elution buffer (10mM Tris, pH8.0)

• • • Method 2.
      • Takes less time and get high yield of products. Templates can be remained.(But it's not affect critically to transformation.)

1. Add 1㎕ of DpnI to not purified PCR products.
2. Incubate at 37℃ about 2~3 hours.
3. This samples were gel-purified and suspended in elution buffer.

Notes

• Primers