(r4) ProtocolsSsdnaRecombination < Lab

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---+ &lambda; 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.

<table width="100%"><tr><td valign="top">
</td><td align="right"><img src="%ATTACHURL%/lego.gif" alt="" /></td></tr></table>

---+++ 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 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 100&mu;M. Therefore ~2164 ng/&mu;L.
      * Working stock is 10&mu;M. Therefore ~216ng/&mu;L
      * Therefore use ~0.5&mu;L
   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 ~100&mu;L 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 &lambda; 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: r4 - 2013-03-27 - DanielDeatherage