Lidstrom:Protocols

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General Lab:

• Health & Safety
• Finding Chemicals
• Autoclave
• Sterile technique
• E. Coli basics
• Preparing Freezer Cell Stocks
• Pouring Media Plates

Cell Culture:

• Tube Spec

DNA/RNA:

DNA

• Intro: Building with DNA (in progress)
• Colony PCR
• Digestion with Restriction Enzymes
• Diluting Primers
• Gibson Assembly (In progress)
• Ligation (in progress)
• Loading Dye Recipe
• Miniprep (in progress)
• Oligo Orders (in progress)
• Overlap Extension PCR (in progress)
• PCR (in progress)
• Purifying DNA (in progress)
• Sequencing with GeneWiz
• Site-Directed Mutagenesis (in progress)
• Transformation (in progress)

E. Coli

• Competent Cell Preparation (in progress)

Electrocompetent Cells

• You can make your own electro-competent cells for electroporation.
  • From Amada's past mentor in undergrad:
    • Grow the cells overnight
    • Inoculate from this the next morning: generally use 200 uL/50 mL
    • Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 3 times with 10% glycerol (everything on ice).
    • Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
      • Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See this page. Would allow you to calculate efficiency.