Molecular Biology

• Culture Media
• Competent Cells
• PCR
• Golden Gate Assembly
• E.coli Transformation
• Glycerol Stocks

• M9 Media
• Plate Reader Fluorescence
• Flow Cytometry

• RNA structure prediction
• Transcriptional Neutralization
• Liquid Handling Automation

Glycerol Stocks

Rationale

Making glycerol stocks is our way of preserving any bacterial strains (plasmid-carrying or not) for the long-term. Stored at -80°C, these stocks will have the bacteria halt their metabolism, to be "re-started" later when we desire.

Glycerol is necessary because water would normally form sharp crystals at -80°C, breaking the cell membrane and killing the cells.

Method

• From 100% glycerol, mix a desired volume with 1 molume of water to make 50% glycerol (it's hard to pipette the 100% accurately)
• Mix 500 uL of 50% glycerol with 500 uL of an overnight bacterial culture to get a 25% glycerol final concentration. Place at -80°C for storage.

Retreiving

To retrieve a culture from a glycerol stock, the recommended method is plating the cells by streaking a small volume of the stock in solid media, then picking a colony to ensure genotype homogeneity (they all have the same DNA), then transferring to liquid culture if needed.

Transfering the stock directly to liquid culture is also possible, assuming the stock is already clonal. However, this leaves opportunity for population heterogeneities to propagate or (in slower-growing bacteria) for contaminations to take over, so proceed with caution.