Overview and materials

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)