Difference: ProtocolsPsltsEditing (2 vs. 3)

Overview

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

Protocol

Materials

• 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
2. 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
   2. 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
   3. 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
   4. 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
3. Gel purify each of the 4 PCR fragments using standard conditions.
4. Mutation Cassette generation using Gibson reaction. 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
   2. Adjust the volume to 10µl with DNase-free water.
   3. Add 10µl of Gibson assembly master mix.
   4. Incubate 1 hr at 50C.
   5. Use 2µl to transform.
   6. Outgrowth 45 minutes at 37C.
   7. Plate overnight on LB crb kan plates.
   8. Verify correct mutation construct using pHA.seq.F and pHA.seq.R primers.

iDT "Gene-block" based

Reference

-- Main.DanielDeatherage - 20 Apr 2015