Caltech/Protocols/Recombineering Protocol
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- | This protocol is adopted almost verbatim from Oppenheim <i>et al</i>, 2004 | + | This protocol is adopted almost verbatim from Oppenheim <i>et al</i>, 2004. |
==Recombineering== | ==Recombineering== | ||
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==References== | ==References== | ||
- | + | # Oppenheim AB, Rattray AJ, Bubunenko M, Thomason LC, and Court DL. ''In vivo recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides''. Virology 2004 Feb 20; 319(2) 185-9. doi:10.1016/j.virol.2003.11.007 pmid:14980479. | |
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Revision as of 22:31, 26 October 2007
iGEM 2007
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This protocol is adopted almost verbatim from Oppenheim et al, 2004. RecombineeringThe strains used for recombineering carry a defective λ prophage containing the pL operon under control of the temperature-sensitive repressor cI857.
Oligonucleotide DesignWe purchased our oligonucleotides from IDT with PAGE purification. The oligos were homologous to the N and Q gene regions to be mutagenized, and contained a single point mutation moving a tyrosine residue to an amber stop codon. The oligo sequences are given below, with the nucleotide introducing the point mutation capitalized. Oligos targeting N for amber mutation5'-tctcctgtcagttagctttggtggtgtgtggcagttCtagtcctgaacgaaaaccccccgcgattggcac-3' 5'-tcaatacgttgcaggttgctttcaatctgtttgtgCtattcagccagcactgtaaggtctatcggattta-3' 5'-ccactgcatgttatgccgcgttcgccaggcttgctCtaccatgtgcgctgattcttgcgctcaatacgtt-3' Oligos targeting Q for amber mutation (courtesy of D. Court)5'-ttagtatttccttcaagctttgccacaccacgCtatttccccgataccttgtgtgcaaattgcatcagat-3' References
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