Heads up to my non-scientist audience: This post is aimed at undergrad/grad school researchers who are already familiar with biology and are trying to understand how a common DNA isolation protocol (called a miniprep) works. That’s why there’s a bit more jargon in this post, which you know I usually try to avoid. Feel free to ask me to clarify or give a non-scientist explanation for anything in this post, or you can just skip this one and enjoy my posts that are aimed at non-scientists.
You’ve done a miniprep or maybe a thousand of them! But you might not know how it works. Each kit is a little different but they all follow the same basic principles.
So let’s talk about what the whole goal is—what are we doing here? You do minipreps to isolate DNA. Not just any DNA—we’re looking for small circular pieces of DNA, called plasmids. Plasmids are incredibly useful tools for biologists! We can use them to put almost any gene we want into the organism we’re studying!
You’ve learned that every organism has a genome, and every cell within the organism has that same genome. Cells read the code of the genome to determine which proteins to make. In addition to the DNA in their chromosomes, bacteria naturally produce these circular pieces of DNA called plasmids. Researchers have taken advantage of the fact that bacteria make these plasmids and carefully manipulated them so that we can now express any gene we want by placing it in a plasmid and transforming it into bacteria.
Once we’ve inserted a plasmid with our gene of interest into bacteria, we can take use rapid bacterial growth to make up huge amounts of the gene we want. Then we explode their cells and extract the plasmid DNA. Once we have the DNA we can do whatever we want with it. For example, we could introduce it into yeast or mammalian cells, or cut out just the portion of this plasmid we want and fuse it to another piece of DNA to make a brand new plasmid.
So, we’re ready to miniprep the bacteria that are living in our culture tube. But how did we get here?
You started with a plate that had a culture of bacteria growing on it. Each colony started as a single cell that grew and divided outward until it became a mass that’s large enough for you to see with the naked eye. You then jabbed a stick into it that has a small number of cells on it, and poked that into a small amount of liquid media in a test tube. That media has lots of nutrients for our bacteria to grow. Now it’s been growing overnight and the few cells you put in have multiplied and multiplied.
Let’s get started! We begin by centrifuging the culture we grew up, using rotational force to pull the heaviest items to the bottom.
Now, you can see that the cells have all been pulled down and the media is at the top. We’ll take off the media and now we just have lots of bacterial cells in our tube.
Now we begin messing with our cells. First, we have to resuspend them into liquid. We use a solution that has
- the sugar glucose to maintain osmotic pressure (so the cells don’t explode or shrivel!)
- the buffering agent Tris to maintain the pH at a moderately basic level, and
- the acid EDTA to weaken the cell envelope. The EDTA will also prevent enzymes called bacterial nucleases from degrading the DNA in later steps.
Next, we’re going to explode the cells! Except, we’re scientists, so we call it lysis. For this, we use a detergent called SDS, which dissolves cellular lipids, including ones in the cell membrane. The solution also has sodium hydroxide which is so basic and thus harsh that all the chromosomal DNA separates into single strands. In contrast, the plasmid DNA is so tightly coiled up that its two strands will be able to stay together.
Next, we’re going to get rid of everything other than the DNA we want, in a process called precipitation. That just means we’re taking something that was dissolved and making it solid again. The solution we use has acetic acid (which you may know as the main ingredient in vinegar) to bring the pH back to neutral. The neutral condition is much less harsh on the DNA, so the chromosomes can become double-stranded again. But the huge pieces of chromosomal DNA can’t come back together neatly as double stranded DNA because of their size, and they get tangled up.
In addition to acetic acid, the precipitation solution also has the salts potassium acetate and guanidium hydrochloride. The columns we use for minipreps have the compound silica in them to bind DNA. Guanidium hydrochloride’s job is to strip water off the silica in the column and off of the DNA. Now that both the silica and the DNA are lonely, they’ll interact with each other. There are also potassium ions in the solution, which form a bridge between the DNA and the silica column. Finally, the solution has the molecule acetate to interact with SDS, lipids, and proteins and pull them out of solution. This whole mess also takes with it the chromosomal DNA, which is all tangled up. The whole thing looks white and goopy.
Next, we do a wash with ethanol. This removes salts as well as any SDS that’s lingering. If we don’t do this, some later applications like restriction enzyme digests won’t work as well.
Finally, we’re going to force our plasmid DNA to move from the silica column above to a solution which will end up coming down into the tube below. We call that eluting. This solution is designed to protect the DNA. Its low salt concentration helps release the DNA from the silica column. The buffering agent Tris maintains the pH at 8, which is just slightly basic. Just like in the solution we used to resuspend our cells, we again use EDTA. EDTA binds magnesium and other ions with a positive charge of +2, so that bacterial nucleases can’t use them to help degrade DNA.
After the solution sits for a few minutes, we do one last spin in the centrifuge and there you have it, your plasmid DNA is in the solution in your microcentrifuge tube!