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
- plasmids
- pKD46 AmpR, 30°C, l-arabinose induced
- pKD3 ChlR, AmpR, 37°C
- pKD4 KanR, AmpR, 37°C
- pCP20 AmpR, ChlR, 30°C
- primers (design specifically for own experiment; order from IDT)
- kits
- Mini-prep
- PCR purify
- Pfx DNA polymerase (enzyme, MgSO4, dNTPs, dH2O, enhancer, buffer, primers)
- 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
- 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.
- equipment
- incubators (30°C,37°C,43°C)
- UV-vis
- electroporator
Basic outline of procedure
- Miniprep all plasmids in host strains
- Transform pKD46 into (chemically competent) target strain, plate out on LB+Amp
- PCR amplify linear fragment from pKD3 or pKD4
- Make {target strain, pKD46}electrocompetent
- Electroporate linear DNA into electrocompetent cells
- Colony PCR to verify antibiotic replacement of gene.
- Get rid of antibiotic resistance
- PCR verify deletion (scar present)
--Heather M. Jensen 14:48, 9 June 2009 (EDT)
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