(r2) Barrick Lab :: ProtocolsSsdnaRecombination

---+ &lambda; 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 &lambda; red electrocompetent cells
   1 Make the line you wish to have your mutation of interest electrocompetent 
      * ProtocolsElectrocompetentCells
   1 Transform &lambda; 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 &lambda; red plasmid) electrocompetent.
      * Must induce &lambda; red proteins before centrifigation.
      * See ProcedureGenomeModificationDatsenkoWanner Day 2 for detailed induction information.

---++ Day 0:
   1 Thaw 50 &mu;L 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 100&mu;M. Therefore ~2164 ng/&mu;L.
      * Working stock is 10&mu;M. Therefore ~216ng/&mu;L
      * Therefore use ~0.5&mu;L


-- Main.DanielDeatherage - 25 Mar 2013
Barrick Lab > ProtocolList > ProtocolsSsdnaRecombination
Contributors to this topic
DanielDeatherage
Topic revision: r2 - 2013-03-25 - 22:01:59 - Main.DanielDeatherage