IGEM:Caltech/2007/Protocols/Recombineering Protocol: Difference between revisions
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==Recombineering== | ==Recombineering== | ||
The strains used for recombineering carry a defective | The strains used for recombineering carry a defective λ prophage containing the pL operon under control of the temperature-sensitive repressor cI857. | ||
# The strain of choice is grown in a shaking water bath at | # The strain of choice is grown in a shaking water bath at 32°C in LB with 0.4% maltose to mid-exponential phase, A600 0.4–0.6 (30 ml is adequate for several recombineering reactions). | ||
# The culture is harvested by centrifugation and resuspended in 1 ml TM (10mM Tris base, 10mM MgSO4, pH 7.4). The phage to be engineered is added at a multiplicity of infection of 1–3 phages/cell (we assume cell density of approximately 10 | # The culture is harvested by centrifugation and resuspended in 1 ml TM (10mM Tris base, 10mM MgSO4, pH 7.4). The phage to be engineered is added at a multiplicity of infection of 1–3 phages/cell (we assume cell density of approximately 10<sup>8</sup>/ml before concentration) and allowed to adsorb at room temperature for 15 min (this step would need modification for other phages, i.e., adsorption on ice). | ||
# Meanwhile, two flasks with 5-ml broth are pre warmed to | # Meanwhile, two flasks with 5-ml broth are pre warmed to 32°C and 42°C in separate shaking water baths. | ||
# The infected culture is divided and half inoculated into each flask; the cultures are incubated an additional 15 min. The | # The infected culture is divided and half inoculated into each flask; the cultures are incubated an additional 15 min. The 42°C heat pulse induces prophage functions; the 32°C uninduced culture is a control. | ||
# After induction, the flasks are well chilled in an ice water bath and the cells transferred to chilled 35-ml centrifuge tubes and harvested by centrifugation at approximately 6500g for 7 min. | # After induction, the flasks are well chilled in an ice water bath and the cells transferred to chilled 35-ml centrifuge tubes and harvested by centrifugation at approximately 6500g for 7 min. | ||
# The cells are then washed once with 30-ml ice-cold sterile water; the pellet is quickly resuspended in 1-ml ice-cold sterile water and pelleted briefly (30 s) in a refrigerated microfuge. | # The cells are then washed once with 30-ml ice-cold sterile water; the pellet is quickly resuspended in 1-ml ice-cold sterile water and pelleted briefly (30 s) in a refrigerated microfuge. | ||
# The pellet is resuspended in 200 | # 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 | # 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 [[IGEM:Caltech/2007/Protocols/Two_Layer|double layer assay]] with amber suppressing and non-suppressing bacteria. | # 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 [[IGEM:Caltech/2007/Protocols/Two_Layer|double layer assay]] with amber suppressing and non-suppressing bacteria. | ||
Latest revision as of 13:39, 26 October 2007
This protocol is adopted almost verbatim from Oppenheim et al, 2004[1]. 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 |