ProtocolsPsltsEditing < Lab

---++ Overview

Lab protocol for using the pSLTS plasmid method of scarless genome editing developed by the Copley lab.

---++Protocol
---+++ Materials
The following general materials should be located before starting:
   * Plasmids pSLTS & pT2SK
   * Primers MF & MR
   * Primers pHAFor & pHARev

---+++ Mutation Cassete Construction
---++++Amplified from genome
   1 Design primers for the mutation cassette. A total of 4 primers must be designed to generate the following (standard primer purification should be sufficient):
      * 5' mutation cassette. A ~200bp fragment containing the mutation of interest in the 30-50bp at the 3' end of the fragment flanked by overhang sequences:
         * 5'-AGGCGTATCACGAGGCCCTTxxxxx where xxxxx represents 14-25bp of homologous DNA at the 5' end of the fragment
         * 5'-ACCGCTGCCACTCTTGAGATxxxxx where xxxxx represents 14-25bp of homologous DNA at the 3' end of the fragment
      * 3' mutation cassette. A ~200bp fragment containing the mutation of interest in the 30-50bp at the 5' end of the fragment flanked by overhang sequences:
         * 5'-GCAGGGCGGGGCGTAAxxxxx where xxxxx represents 14-25bp of homologous DNA at the 5' end of the fragment
         * 5'-CTCACATGTTCTTTCCTGCGxxxxx where xxxxx represents 14-25bp of homologous DNA at the 3' end of the fragment
   1 PCR amplify the following:
      1 Plasmid backbone. (May be available as lab stock as this is not specific to any project)
         * Template: pT2SK
         * Primers: pHAFor & pHARev
         * Conditions:
         * Expected size: 1887
      1 Selection cassette. (May be available as lab stock as this is not specific to any project)
         * Template: pT2SK
         * Primers: MF & MR
         * Conditions:
         * Expected size: 1217
      1 5' mutation cassette.
         * Template: Genomic DNA purified from strain of interest containing mutation you wish to introduce into new strain.
         * Primers: Primers designed in previous step for 5' mutation cassette
         * Conditions: Will vary.
         * Expected size: ~200bp depending on specific fragments
      1 3' mutation cassette.
         * Template: Genomic DNA purified from strain of interest containing mutation you wish to introduce into new strain.
         * Primers: Primers designed in previous step for 5' mutation cassette
         * Conditions: Will vary.
         * Expected size: ~200bp depending on specific fragments
   1 Gel purify each of the 4 PCR fragments using standard conditions.
   1 Mutation Cassette generation using Gibson reaction. [[ProtocolsGibsonCloning][General Gibson Reaction Protocol]]
      1 Mix the following products at the given amount:
         * Plasmid Backbone: 25 fmol
         * Selection Cassette: 75 fmol
         * 5' Mutation Cassette: 125 fmol
         * 3' Mutation Cassette: 125 fmol
      1 Adjust the volume to 10µl with DNase-free water.
      1 Add 10µl of Gibson assembly master mix.
      1 Incubate 1 hr at 50C.
      1 Use 2µl to transform.
      1 Outgrowth 45 minutes at 37C.
      1 Plate overnight on LB crb kan plates.
      1 Verify correct mutation construct using pHA.seq.F and pHA.seq.R primers.



---++++iDT "Gene-block" based

---++Reference
[[http://www.biomedcentral.com/1472-6750/14/84][Kim et al BMC Biotechnol. 2014 Sep 25;14(1):84.]]



-- Main.DanielDeatherage - 20 Apr 2015
This topic: Lab > WebHome > ProtocolList > ProtocolsPsltsEditing
Topic revision: r2 - 2015-04-21 - DanielDeatherage