NanoBio: Overview and Materials for λ-red

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
 * 2) *pKD46 AmpR, 30°C, l-arabinose induced
 * 3) *pKD3 ChlR, AmpR, 37°C
 * 4) *pKD4 KanR, AmpR, 37°C
 * 5) *pCP20 AmpR, ChlR, 30°C
 * 6) primers (design specifically for own experiment; order from IDT)
 * 7) kits
 * 8) *Mini-prep
 * 9) *PCR purify
 * 10) *Pfx DNA polymerase (enzyme, MgSO4, dNTPs, dH2O, enhancer, buffer, primers)
 * 11) reagents
 * 12) *If available, chemically competent cells from cell line for knockout
 * LB
 * 1) *Amp, Chl, Kan freezer stocks
 * 2) *Sterile, cold 10% glycerol
 * 3) *Sterile, cold ddH2O
 * 4) *Sterile 1M L-arabinose
 * 5) LB plates
 * 6) *LB alone
 * 7) *LB+Kan (5μg/mL) for "Lo" concentration plates
 * 8) *LB+Chl (5μg/mL) for "Lo" concentration plates
 * 9) *LB+Amp
 * 10) *Also, LB+Kan and LB+Chl at normal concentrations.
 * 11) equipment
 * 12) *incubators (30°C,37°C,43°C)
 * 13) *UV-vis
 * 14) *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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