E. coli knockout

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Revision as of 08:05, 2 August 2010

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