E. coli knockout

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Revision as of 07:48, 2 August 2010 (view source) Csbl (Talk | contribs) (→Procedure) ← Older edit		Revision as of 08:00, 2 August 2010 (view source) Csbl (Talk | contribs) (→Procedure) Newer edit →
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	***Sequences from cassette region have about 60℃ Tm-value.		***Sequences from cassette region have about 60℃ Tm-value.
	***Overview is attached.  [[media:overview.jpg|File:Overview]]		***Overview is attached.  [[media:overview.jpg|File:Overview]]
-	*PCR condition	+	**PCR condition
-	** Anneling temparature and extension time is depend on the target sequence.	+	*** Anneling temparature and extension time is depend on the target sequence.
-	** Anneling temparature is tested at 55℃ at first.	+	*** Anneling temparature is tested at 55℃ at first.
-	** Extension time is depend on the length of the target sequence. (1kb = about 1 min.)	+	*** Extension time is depend on the length of the target sequence. (1kb = about 1 min.)
-	** Detail conditions are shown below.[[Image:PCR condition.jpg|center|thumb|400px]]	+	*** Detail conditions are shown below.[[Image:PCR condition.jpg|center|thumb|400px]]
		+	**Treatment of PCR products
		+	***Method 1.
		+	# PCR products were gel-purified.
		+	# Digested with DpnI for 2~3 hours.
		+	# 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.
		+	# Add 1㎕ of DpnI to not purified PCR products.
		+	# Incubate at 37℃ about 2~3 hours.
		+	# 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 08:00, 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.

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)

• • • • Takes more time and product concentration is lower than method 2. But template vectors remain less.
    • Method 2.

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.

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

Notes

• Primers