First, I had to thaw out and mix the E. coli DNA and the plasmids together in a single tube, just like I was mixing waffle mix and milk in a bowl to make pancake batter.
Immediately after mixing my E. coli and plasmids together, I cold-shocked them by putting their tube in ice for exactly two minutes, immediately put them in a hot water bath for exactly 30 seconds afterwards, and returned them to the ice for exactly two minutes following the hot bath.
After that, with much less urgency, I added a handful of buffer solutions to my E. coli + Plasmid solution to stabilize the shocked DNA, then spun them really fast and hard in a centrifuge to separate the junk from the DNA I wanted. The stabilized DNA (which included the plasmids and the still-living E. coli cells) floated to the top of the tube, while the unwanted junk (such as dead E. coli cells and excess buffer solutions) formed a nasty little pellet at the bottom.
I transferred the less-dense liquid containing my wanted stuff into a new vial, used a couple different wash buffers to purify the solution, then added a sugary, nutrient-dense goo to whatever was left of my living, hopefully-transformed E. coli cells.
Finally, I added that sugary, gooey mixture of transformed E. coli cells to three different agar plates to grow cultures of the stuff. One agar plate simply contained the agar (which is just a fancy term for unflavored Jell-O) for anything and everything to grow on. One agar plate was laced with an antibiotic called Kanamycin, which was pretty much guaranteed to kill everything that touched its plate. The last agar plate was laced with Ampicillin, upon which only my transformed E. coli cells would grow upon.
All three of those agar plates were put into an incubator to grow for two days.
While having multiple plates may seem redundant and even wasteful, each plate served a specific, important purpose.
The first plate- the one that wasn’t laced with any antibiotics- was my “positive control”, meaning that unless something went horribly wrong, something was guaranteed to grow on the plate. The second plate was my negative control, meaning that unless something went horribly wrong, nothing was gonna grow on that plate. The third and final plate was my actual experimental plate- the plate that would only grow the E. coli that I wanted it to grow- which was my successfully-transformed, glow-in-the-dark, Ampicillin-resistant E. coli.
To be continued...
