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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# The pellet is resuspended in 200 μL cold sterile water and 50–100 μl aliquots are used for electroporation with 100–150 ng PCR product or 10–100 ng oligonucleotide. (Oppenheim <i>et al</i> use a BioRad E. coli Gene Pulser set at 1.8 mV with 0.1-cm cuvettes.) | # The pellet is resuspended in 200 μL cold sterile water and 50–100 μl aliquots are used for electroporation with 100–150 ng PCR product or 10–100 ng oligonucleotide. (Oppenheim <i>et al</i> use a BioRad E. coli Gene Pulser set at 1.8 mV with 0.1-cm cuvettes.) | ||
# Electroporated cells are diluted into 5 ml 39°C LB medium and incubated to allow completion of the lytic cycle. | # Electroporated cells are diluted into 5 ml 39°C LB medium and incubated to allow completion of the lytic cycle. | ||
- | # The resulting phage lysate is diluted and titered on appropriate bacteria to obtain single plaques. To screen for phages containing desired amber mutations in <i>N</i> and <i>Q</i>, the lysate can be titered in a [[ | + | # The resulting phage lysate is diluted and titered on appropriate bacteria to obtain single plaques. To screen for phages containing desired amber mutations in <i>N</i> and <i>Q</i>, the lysate can be titered in a [[Caltech/Protocols/Two_Layer|double layer assay]] with amber suppressing and non-suppressing bacteria. |
==Oligonucleotide Design== | ==Oligonucleotide Design== | ||
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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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Latest revision as of 22:32, 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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