Cloning protocols

• Cloning Workflow Overview
  • Step 1: Generate DNA fragments
  • Step 2: Assemble plasmids
  • Step 3: Transform bacteria
  • Step 4: Screen colonies
  • Step 5: Purify plasmid DNA
  • Step 6: Sequence
  • Step 7: Glycerol stock
  • Starting with an assembled plasmid
• Cloning Workflow Timeline
  • Typical Timeline
  • Accelerated Timeline
• Colony PCR
  • Materials
  • Protocol
  • For hard to amplify templates
• Restriction digest
• Gel electrophoresis and extraction of DNA
  • Gel electrophoresis
  • Gel extraction
• Gibson assembly
  • Protocol
• Glycerol Stocking
• Golden Gate assembly
  • Protocol for PCR fragments
  • Protocol for pPV plasmids
  • Reference for pPV connector sequences
  • Reference for entire Golden Gate workflow
• Ligation assembly
  • Protocol
• LR cloning
  • Protocol
  • Expected results
• Oligo annealing for ligation cloning
  • Oligo phosphorylation
  • Oligo annealing
  • Combined oligo phosphorylation and annealing
• PCR
  • Q5
  • PrimeSTAR
  • Taq
  • KOD Xtreme
  • Confirm and purify
• Plasmid Prepping
  • Miniprep
  • Midiprep
  • Maxiprep
• Cloning tips, tricks, and troubleshooting
  • PCRs
  • Gibsons
  • Golden Gates
  • Generally difficult assemblies
• Touchdown PCR
  • Touchdown PCR
• Bacterial Transformation
  • Cloning Summary
  • Transformation (Chemically Competent Bacteria)