---+ λ red mediated ssDNA gene modification ---++ Background Protocol designed based on 3/16/11 Court Lab Protocol ([[http://redrecombineering.ncifcrf.gov/Protocols_files/step-by-step%20ssDNA%20protocol.pdf which is available here]]) with Barrick lab specific modifications. Modifications include: * Detailed description of primer design * Protocol streamlined for 2 electroporations for single base mutation ---++ Before Starting ---+++ Design/Order Oligos * A minimum of 6 oligos must be ordered for each mutation. (2 mutation oligos, 2 screening oligos, 1 sequencing oligo, and 1 common oligo) 1 Mutation Oligos: * 2 different 71bp oligos will be needed for each mutation. Oligo 2 will have your mutation with 35bp of background on each side, and Oligo 1 with that mutation plus 2 bp mutated to random sequence on each side of your mutation. * Following examples based on 1bp SNP. b = background, __M__ = mutation of interest, *R* = Random mutation, can be any single base substitution, but not degenerate. * Mutation Oligo 2: bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb bb __M__ bb bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb * Mutation Oligo 1: bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb *RR* __M__ *RR* bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb * Mutations __MUST__ be placed on the lagging strand. * For _E. coli_ REL606 (and other B strains) origin of replication is at ~10:00 not 12:00 (3886105-3886336). Therefor: * If mutation location between ~ 3,886,500 and ~ 1,571,300 : design against reverse strand * If mutation location between ~ 1,572,000 and ~ 3,886,000 : design against forward strand * If mutation location between ~ 3,886,000 and 3,886,500 or ~1,571,300 and 1,572,000 it's probably best to try both strands and see which is better. Expect 100+ fold increase in efficancy for lagging strand. 1 Mutation Screen Oligo: * Primers used for screening colonies for presence of transformants * Should be ~20bp long, amplicon of ~150<sup>+</sup> bp, and one primer must have 3' bases as mutations of interest. * Mutation Screen 2: bbbbbbbbbbbbbbb bb __M__ bb * Mutation Screen 1: bbbbbbbbbbbbbbb *RR* __M__ *RR* * Both screen's can be done with a common primer at least 150 bp away on the opposite strand. * Primer can also be used for sequencing oligo 1 Mutation Sequencing Oligo: * Primer to be used for final sequencing to verify mutation on correct background. * Primer should be ~20bp long, ~150<sup>+</sup> bp upstream of Mutation screen oligo. ---+++ Make λ red electrocompetent cells 1 Make the line you wish to have your mutation of interest electrocompetent * ProtocolsElectrocompetentCells 1 Transform λ red plasmid into cells and select with appropriate antibiotic. * pKD46 = Amp<sup>R</sup> & 32<sup>+</sup> temperature sensitive replication * pKD78 = Cam<sup>R</sup> & 32<sup>+</sup> temperature sensitive replication * ProtocolsElectrocompetentCells Bottom section, but outgrowth __MUST__ be done at 30C 1 Make new line (background of interest with λ red plasmid) electrocompetent. * Must induce λ red proteins before centrifigation. * See ProcedureGenomeModificationDatsenkoWanner Day 2 for detailed induction information. ---++ Day 0: 1 Thaw 50 μL aliquot of electrocompetent cells on ice. 1 Transfer cells to electroporation cuvette. 1 Add ~100ng of Mutation Oligo 1 to pre-chilled cuvette. * M.W. of ssDNA = (# nucleotides x 303.7) + 79.0. Therefore assuming 71 nucleotides, MW ~= 21642.41. * Typical dilution of oligos is 100μM. Therefore ~2164 ng/μL. * Working stock is 10μM. Therefore ~216ng/μL * Therefore use ~0.5μL 1 Pipette gently but thoroughly * Do not vortex * Use filter tips if working doing multiple mutations at once 1 Electroporate, and immediately add 1ml of room temperature LB to cuvette. * If preparing multiple samples, add LB before doing next electroporation. * Cuvette with LB can be left at room temperature while rest of electroporations are finished. 1 Transfer all liquid from cuvette to sterile tube for 30C outgrowth. 1 If no selectable phenotype, 30 minutes is best. * Explanation: Cells typically have 4-8 copies of the chromosome per cell when recombination takes place, but recombination will typically only take place on a single strand. 30 minutes of outgrowth allows the cells to recover, but chromosomes will not yet have segregated. Colonies will therefore be a mixture of recombinant and parental cells, with percentage of recombinant cells being between ~25% and 12.5%. Colonies that initially test present for transformants can then be streaked for single colonies which will be found at between 25 and 12.5%. Comparatively if chromosomes allowed to segregate, 4 to 8 times more colonies will need to be screened to find a single transformant. 1 If mutation causes selectable phenotype, more than 2 hours of outgrowth should be performed in 10mL LB. 1 Dilute cells 1x10<sup>-4</sup> to 1x10<sup>-6</sup>, and plate ~100μL on LB + antibiotic 1 Allow plate to incubate overnight at 30C. * Warning: lambda red plasmid is temperature sensitive above 32. ---++ Day 1: Initial PCR Screening 1 PCR with Mutation Screen Oligo 1 and Common oligo. * Because the 3' mutation screen oligo will only bind to DNA that has been mutated, presence of a band signifies recombination. * Likely that the brighter the band, the better candidate (ie greater percentage of cells recombinants). 1 30 cycles of standard colony PCR conditions acceptable. 1 Initially test 40 colonies is recommended. 1 Be sure to either patch cells or store in small amount of saline. * If cells patched, allow to grow overnight and re-plate for single cells following day. 1 Positive 2-3 positive colonies should be spread for single cells and incubated overnight at 30C on LB with antibiotic. ---++ Day 2: Confirmation Screening 1 Single colonies from plates should be subjected to the same protocol as was used on Day 1. * __Need experimental validation: 20-30 colonies should be sufficient to screen.__ 1 Positive colonies should be used to inoculate LB+antibiotic overnight 30C cultures. * 1 or 2 colonies should be sufficient 1 Positive colonies can be PCR'd with common primer and sequencing primer and sent for sequencing off either primer if desired. * Should not be necessary under normal circumstances. ---++ Day 3: Fixing 4 bp mutation 1 Make freeze down of overnight culture, this is an intermediate with a 5bp mutation. 1 Make cells electrocompetant as previously being sure to induce the λ red proteins. 1 Electroporate as in Day 0, but use Mutation Oligo 2. ---++ Day 4: Background screen 1 Repeat Day 1 protocol using Mutation Screen oligo 2 rather than 1. ---++ Day 5: Final screen and sequencing 1 Repeat Day 2 protocol. 1 Positive colonies should be used to inoculate o/n cultures. These should contain the correct single base mutation. 1 Additional sequencing PCR needs to be preformed. This should be using common primer and sequencing primer. 1 Submit sample for sequencing with either primer. -- Main.DanielDeatherage - 25 Mar 2013
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Topic revision: r3 - 2013-03-26 - DanielDeatherage