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.
  2. Mutation Screen Oligo:
    • Primers used for screening colonies for presence of transformants
    • Should be ~20bp long, amplicon of ~150+ 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
  3. Mutation Sequencing Oligo:
    • Primer to be used for final sequencing to verify mutation on correct background.
    • Primer should be ~20bp long, ~150+ 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
  2. Transform λ red plasmid into cells and select with appropriate antibiotic.
    • pKD46 = AmpR & 32+ temperature sensitive replication
    • pKD78 = CamR & 32+ temperature sensitive replication
    • ProtocolsElectrocompetentCells Bottom section, but outgrowth MUST be done at 30C
  3. 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.
  2. Transfer cells to electroporation cuvette.
  3. 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 - 25 Mar 2013 - 22:01:59 - Main.DanielDeatherage
 
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