---+ λ red mediated ssDNA gene modification ---++ Background Protocol designed based on 3/16/11 Court Lab Protocol (**hyperlink or attachment to/of protocol**). With Barrick lab specific modifications. ---++ Before Starting ---+++ Design/Order Oligos * A minimum of 6 oligos must be ordered for each mutation. (2 mutaiton 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 substituion, 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_ 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 laggin 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. * bbbbbbbbbbbbbbb bb __M__ bb * 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 alliquot of electrocompetent cells on ice. 1 Transfer cells to electroporation cuvette. 1 Add ~100ng of Mutation Oligo 1 to 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 -- Main.DanielDeatherage - 25 Mar 2013
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Topic revision: r2 - 2013-03-25 - DanielDeatherage