This is a step-by-step hand holding, descriptive, illustrated picture guide to extract a plasmid contain a green fluorescent protein (GFP) gene from Escherichia coli (E. Coli) we’ve grown.

This is a continuation of Bio-Quest #1, where we’ve grown colonies of bacteria containing a plasmid that has both a GFP gene and ampicillin resistance gene. While in the last quest we took GFP from stock source and transformed E. Coli to grow on plates, we’re now going to take our grown E. Coli, incubate it at a higher volume in liquid media, and then extract a high volume of plasmids containing the GFP gene. In a sense, we’re creating our own GFP plasmid stock from a mass of E. Coli incubated in some media overnight.

Time / Scheduling

Start → End: 2 Days (E Coli. growth happens overnight)

Focused Lab Work: 4 Hours

Steps

Day 0) Prep

1. Plan. How much ng/ml of plasmid do we want to extract?
2. Check Materials

Day 1)

1. Create “master mix” of LB Broth + Ampicillin
2. Extract colonies from plates via toothpick and place in tubes (1 ctrl, 2 col)
3. Incubate (12-16 hr)
4. Clean up, sterilizing workspace + re-autoclaving toothpicks

Day 2) Plasmid Prep/Extract

1. Centrifuge 1.5ml LB broth with e. coli for cells, toss out supernatant (aka residual liquid)
2. “Re-Suspend” e. coli cells (pellet broken up into buffer)
3. Break Cell Walls/Membranes (aka Lyse) in buffer