Do you want to omit the in vitro #recombination step in your #SDM? Well, #transformation into a cell with #Phage recombination machinery can help you with that through #REPLACR (Recombineering of Ends of linearised PLAsmids after PCR). REPLACR employs #HomologousRecombination between #PCRamplicon ends to generate circular plasmids with intended mutation(s).
Principle
#Recombineering is #Recombination mediated #GeneticEngineering where specific recombination proteins are put to work instead of the traditional #RestrictionEndonuclease and #Ligase based approach. #REPLACR works by transforming #SDMPCR products with #homologous ends into competent bacteria expressing #PhageRecombinationProteins. These proteins help the #SDMPCR products recombine and form the mutant plasmid. In this case #Red/ET recombineering system (Red γ, β, α and RecA) containing recombineering plasmid (#pSC101BADgbaRecA[tet]) with #HS996 E.coli was used.
Protocol
Primer design: A good graphical representation of #REPLACRprimer design approaches is shown above. In this case the #homology region of 17 bases has been shown to work the best in primers of length 23 bases.
Electrocompetent cell preparation: Please refer to the paper.
PCR and ethanol precipitation: PCR was done with high fidelity #KOD-Xtreme enzyme. #Ethanol precipitation is an optional step.
DpnI digestion: 1-2μL of #DpnI added in the #PCR product and incubated at 37C
Transformation/ electroporation: 100ng #PCR product was added to 50μL of electrocompetent cell in a 1mm cuvette, and pulsed at at 1.35 kV, 25 μF, 200 ohms. With recovery in LB for 1-2 hours, cells were plated and incubated in 37C.
Colony screening and clone selection.
Merits
Single PCR based approach, #quicker.
#Efficiency is 84% ( median is 75% for all kinds of modifications)
#Tremendous deletion range (~144kb deletion achieved in #BAC)
Demerits
Lab-made electro-competent cells required.
For insertion of >60bp, primer synthesis is expensive.
More to dig into:
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