- To calculate an exact recipe for digesting a specific piece of DNA with a specific set of enzymes.
- To sketch out the practical considerations to keep in mind when designing a DNA digest.
- Star activity
Hi everybody, welcome back to Synthetic Biology One. Today we’re going to talk about digesting DNA: cutting it up into pieces using enzymes. This is something that we do all the time in the lab. But the exact protocol changes day to day depending on the application.
Working in the lab is a little bit like cooking. As a beginner, you follow the recipe exactly. Gradually, you learn that some parts of the recipe can be changed, while other changes ruin the food. One day you wake up and you’re a master chef, and you can create your own recipes. Today will become a master chef DNA digests.
Let’s start by looking at the ingredients of a typical digest
The DNA is there to be digested. The main thing we need to know about it is the concentration. A typical sample of DNA that we use in the lab might be 100 ng/μl. But a wide range is possible. Your sample might be more or less concentrated by a factor of 10, and generally that is no problem.
The enzymes are there to do the digesting. For these we need to know the activity, which you can find in the documentation from the manufacturer. A typical activity might be 10 U/μl, which means that 1 μl of enzyme can digest about 10 μlg of DNA in 1 hour.
The reaction buffer creates the ideal conditions for the enzymes to act. The right pH, the right magnesium concentration and so on. Wherever you buy your enzymes, they will always come with appropriate reaction buffer. A common mistake is thinking that reaction buffers are interchangeable. But they are not. So don’t be a maverick, you follow the manufacturers instructions.
We add additional water to control the total reaction volume. In this way, we can control the concentration of all our other ingredients.
To set up a digest, we need to choose the right amounts for each of these 4 ingredients. Let’s work backwards to see how to think about these choices.
First Question: What are the limits on the total reaction volume? In general, I like large reactions. But you can’t just fill your bathtub with DNA, there are limits on how big your digests can be. For example, if you are planning to load your digest into a gel, a typical well can only hold about 50 μl. Of. Of you are loading your digest into a DNA purification column, these can only take about 300 μl. Above that, you have to consider that your Eppendorf tubes can only hold 1 or 2 ml. Above that, then what are you trying to do you crazy person? Why do you need that much DNA?
The total reaction volume dictates the volume of reaction buffer. These buffers are always supplied at 10x concentration. That means in a 50 μl reaction, you will be using 5 μl of buffer. Easy.
The reaction volume also sets a limit on the amount of enzyme you can use. Here is the thing: restriction enzymes are supplied in 50% glycerol. Glycerol stabilizes the enzymes in the freezer. But it also interferes with the ability of the enzymes to cut effectively. As a rule of thumb, enzymes get messed up at about 5% glycerol concentration. They start cutting nonspecifically, just all over the place, in what is called star activity.
Your enzymes should be diluted by at least a factor of 10 in your final reaction. So to a 50 μl reaction, we can only add a maximum of 5 μl of enzymes. I like to come in under that, just to be safe. Let’s say 1 μl. Noting will ruin your day, or your DNA, like star activity.
Now that we know how much enzyme we can add, we know the limits on our DNA concentration. In principle, 1 μl of enzyme gives us 10 units of activity, enough to digest 10 μg of DNA in one hour. I like to be conservative here, and add much less DNA than the enzyme can handle. That way I’m covered in case the enzyme is old, or maybe not as amazing at the seller claims. But even 1 μg of DNA is a ton of DNA for most applications.
For example, maybe you are doing an analytical digest and you just need to see your DNA on a gel. For this, 10 ng is plenty. If your goal is to purify this DNA and use it in cloning, then you probably want a little more. 1 μg is enough for 99% of what we do in this lab on a normal day.
So now we’ve built up our recipe, we’ll put our samples in a 37 C incubator and leave them for an hour or more. But for how long exactly? We still have one more question. How important is it that your reaction is goes to completion? Do you really need to cut up every last piece of DNA? Or is a little bit of uncut stuff OK. See here’s the thing. The first 99% of the DNA is easy for the enzymes to find and cut. But as the concentration of uncut DNA gets lower and lower, the reaction rate slows down. Those last few pieces of DNA take a long time to get.
If you’re just running a gel, then you don’t care. You won’t see the 1% of the fragments anyway. But if you’re going to use your sample for a PCR, then you can easily amplify those undigested fragments. If you’re going to transform your sample, you really need to be sure to get all those last few pieces. That means using much less DNA than the enzyme can handle and letting the reaction incubate for longer. Two, three, or even 4 hours to get everything.
And… voila. You are now a master chef of DNA digests. With a little practice, your DNA digests will come out delicious. Just kidding you should never eat in the lab. Ok until next time, happy digesting.
The gel without fluorescent illumination:
The gel with the fluorescent lights on:
The gel with interesting features marked: