Protocols

Our lab has published several protocols on recombineering. We strongly encourage you to consult several of them. Depending on your exact needs, parts of protocols will need to be mixed and matched. At the bottom of this page, you can also download some more general protocols we use in the lab.


General Protocols

    Thomason, L. C., Sawitzke, J. A., Li, X., Costantino, N. & Court, D. L. (2014). Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol 106, 1 16 1-1 16 39. CPMB 2014.pdf.

    Sawitzke, J. A., Thomason, L. C., Costantino, N., Bubunenko, M., Datta, S. & Court, D. L. (2007). Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol. 421, 171-199. MIE.pdf

       

Recombineering with ssDNA Oligos

    Sawitzke, J. A., Costantino, N., Li, X. T., Thomason, L. C., Bubunenko, M., Court, C. & Court, D. L. (2011). Probing Cellular Processes with Oligo-Mediated Recombination and Using the Knowledge Gained to Optimize Recombineering. J. Mol. Biol. Oligo optimization.pdf


Recombineering to BACs

    Sharan, S. K., Thomason, L. C., Kuznetsov, S. G. & Court, D. L. (2009). Recombineering: a homologous recombination-based method of genetic engineering. Nat. Prot. 4, 206-223. BACS.pdf

    Chan, W., Costantino, N., Li, R., Lee, S.C., Su, Q., Melvin, D., Court, D.L., and Liu, P. (2007). A recombineering based approach for high-throughput conditional knockout targeting vector construction. Nucleic Acids Res 35, e64. high throughput.pdf


Recombineering to Plasmids

    Thomason, L. C., Costantino, N., Shaw, D. V. & Court, D. L. (2007). Multicopy plasmid modification with phage λ Red recombineering. Plasmid 58, 148-158. plasmids.pdf


Recombineering to Phage

    Thomason, L. C., Oppenheim, A. B. & Court, D. L. (2009). Modifying bacteriophage lambda with recombineering. Methods Mol Biol 501, 239-51.phage.pdf


We also recommend the following reference for an excellent method for recombineering phage:

        Marinelli, L. J., Piuri, M., Swigonova, Z., Balachandran, A., Oldfield, L. M., van Kessel, J. C. & Hatfull, G. F. (2008). BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS ONE 3, e3957.


Protocols available for download


Click on the protocols below to download a PDF of that protocol. The date the protocol was last updated is as listed.


 Recombineering competent cells.pdf 2/8/11

 transformation by electroporation.pdf 2/18/11

 step-by-step ssDNA protocol.pdf 3/16/11

 Brief ssDNA protocol.pdf 3/22/11

 step-by-step knockout (dsDNA) protocol.pdf 3/21/11

 brief dsDNA.pdf 3/14/11

 checklist for plasmid recombineering.pdf  3/14/11


General Bacterial Genetics Protocols

PI Transduction: Thomason-2007 P1 prot.pdf





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