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 = Amp^R & 32⁺ temperature sensitive replication
   • pKD78 = Cam^R & 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