NanoBio: Overview and Materials for λ-red

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Overview

This is λ red gene-knockout protocol that our lab uses. It is adapted from the Datsenko and Wanner paper (PNAS, 2000). The goal of this protocol is to use PCR and a plasmid coding for recombinase proteins (pKD46) to knockout the gene-of-interest and replace it with an antibiotic resistance gene. The antibiotic resistance can then be removed by a second plasmid (pCP20).

Materials

  1. plasmids
    • pKD46 AmpR, 30°C, l-arabinose induced
    • pKD3 ChlR, AmpR, 37°C
    • pKD4 KanR, AmpR, 37°C
    • pCP20 AmpR, ChlR, 30°C
  2. primers (design specifically for own experiment; order from IDT)
  3. kits
    • Mini-prep
    • PCR purify
    • Pfx DNA polymerase (enzyme, MgSO4, dNTPs, dH2O, enhancer, buffer, primers)
  4. reagents
    • If available, chemically competent cells from cell line for knockout
    • LB
    • Amp, Chl, Kan freezer stocks
    • Sterile, cold 10% glycerol
    • Sterile, cold ddH2O
    • Sterile 1M L-arabinose
  5. LB plates
    • LB alone
    • LB+Kan (5μg/mL) for "Lo" concentration plates
    • LB+Chl (5μg/mL) for "Lo" concentration plates
    • LB+Amp
    • Also, LB+Kan and LB+Chl at normal concentrations.
  6. equipment
    • incubators (30°C,37°C,43°C)
    • UV-vis
    • electroporator

Basic outline of procedure

  1. Miniprep all plasmids in host strains
  2. Transform pKD46 into (chemically competent) target strain, plate out on LB+Amp
  3. PCR amplify linear fragment from pKD3 or pKD4
  4. Make {target strain, pKD46}electrocompetent
  5. Electroporate linear DNA into electrocompetent cells
  6. Colony PCR to verify antibiotic replacement of gene.
  7. Get rid of antibiotic resistance
  8. PCR verify deletion (scar present)


--Heather M. Jensen 14:48, 9 June 2009 (EDT)

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