Team:Harvard/Lambda Red
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Lambda red recombineering makes use of homologous recombination systems to allow the insertion of constructs into the genome. It is accomplished in two steps, as shown in the diagram below and in our [https://2011.igem.org/Team:Harvard/Protocols#Lambda_Red Protocols] section. See [https://2011.igem.org/Team:Harvard/Results/One-Hybrid_Selection#Building_the_selection_strain:_Lambda_Red_Recombineering Lambda Red results] section for how we used lambda red to build our selection system. | Lambda red recombineering makes use of homologous recombination systems to allow the insertion of constructs into the genome. It is accomplished in two steps, as shown in the diagram below and in our [https://2011.igem.org/Team:Harvard/Protocols#Lambda_Red Protocols] section. See [https://2011.igem.org/Team:Harvard/Results/One-Hybrid_Selection#Building_the_selection_strain:_Lambda_Red_Recombineering Lambda Red results] section for how we used lambda red to build our selection system. | ||
- | + | ==How to use lambda red== | |
- | For efficient genome editing using lambda red, you can use [https://2011.igem.org/Team:Harvard/Results/Biobricks#EcNR2_strain_.28BBa_K615002.29 the ECNR2 strain] with your own insertion construct and overhangs (i.e. 30-50bp homology to the locus in which the gene is being inserted). To obtain this strain, you can make a request at [http://partsregistry.org/Part:BBa_K615002 the Registry of Standard Biological Parts.] | + | For efficient genome editing using lambda red, you can use [https://2011.igem.org/Team:Harvard/Results/Biobricks#EcNR2_strain_.28BBa_K615002.29 the ECNR2 strain] with your own insertion construct and overhangs (i.e. 30-50bp homology to the locus in which the gene is being inserted). The ECNR2 strain is especially suited for lambda red recombineering, because in addition to having the lambda-phage based recombination system, it also has the mutS gene knocked out to reduce DNA mismatch repair activity, so that the insert (which will not match the original genomic code) is less likely to be excised. To obtain this strain, you can make a request at [http://partsregistry.org/Part:BBa_K615002 the Registry of Standard Biological Parts.] |
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Revision as of 03:24, 29 September 2011
Overview | MAGE | Chip-Based Synthesis | Lambda Red | Protocols
Lambda Red
Lambda red recombineering makes use of homologous recombination systems to allow the insertion of constructs into the genome. It is accomplished in two steps, as shown in the diagram below and in our Protocols section. See Lambda Red results section for how we used lambda red to build our selection system.
How to use lambda red
For efficient genome editing using lambda red, you can use the ECNR2 strain with your own insertion construct and overhangs (i.e. 30-50bp homology to the locus in which the gene is being inserted). The ECNR2 strain is especially suited for lambda red recombineering, because in addition to having the lambda-phage based recombination system, it also has the mutS gene knocked out to reduce DNA mismatch repair activity, so that the insert (which will not match the original genomic code) is less likely to be excised. To obtain this strain, you can make a request at the Registry of Standard Biological Parts.
Figure 1. How to perform lambda red. First, we run a PCR to get the required insertion product (zeocin in this example), and then use lambda red recombination to insert the desired product into the genome. 915px
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