#2: Setting up a home lab and doing a DNA Extraction Kit

#2: Setting up a home lab and doing a DNA Extraction Kit

#2: Setting up a home lab and doing a DNA Extraction Kit

By Rohan M.

Strawberry-DNA header image by Rohan & DALL·E Open AI

So, your DNA Playground just arrived in the mail and you can't wait to get started! Now, I'm just as ready to engineer some bacteria as you are, but there are a few housekeeping items we have to get out of the way first. While the trope of the mad scientist is something that the most dramatic of us (yes, guilty...) tend to romanticize, you really don't want to stray into any legal gray area with your experiments. Luckily, all the experiments we'll be doing are classified as Biosafety Level 1 (BSL-1) or Risk Group 1 (RG-1). Biosafety Levels and Risk Groups are a way of measuring how dangerous a given experiment is, on a scale from 1 (least dangerous) to 4 (most dangerous). 

Citizen scientists like you and me will operate at BSL-1 99% of the time, as it relies on non-pathogenic and relatively harmless microorganisms. That said, if you or people close to your experiment are immunocompromised, you should always talk to a doctor before working with living organisms! In North America, RG-1 experiments can be done without government approval in homes, schools, and makerspaces. However, if you live outside of North America, you'll want to check what your country's policies are regarding RG-1 experiments and genetic engineering before deciding where to set up shop. For more information on biosafety, you can have a look at Chapter 2 of Zero to Genetic Engineering Hero, or check out Amino Labs’ biosafety blog post

Ok, now you can unbox your Playground (I'll pretend that you weren't "multitasking" while half-listening to my extremely valuable advice). You'll probably notice that it has quite a bit of heft. That's because it's a three-in-one machine. It replaces the heating, cooling, and incubation units that are usually needed when working with bacteria or other living organisms. It's hard to appreciate just how amazing this is if you've never had to prepare your own ice buckets and hot water baths before, but trust me when I say that for those of us without the room to accommodate a DIY biology lab – so, you know, ~99% of the population – it's a godsend. 

The DNA playground is a three-in-one machine that allows you to instantly set up a hot station, cold station, and incubator right in your school, home, or local maker space!

The DNA playground is a three-in-one machine that allows you to instantly set up a hot station, cold station, and incubator right in your school, home, or local maker space!

 

 

There are a few key criteria you'll want to keep in mind when deciding where to place your Playground. A hard, non-porous surface and floor are number one. This will allow both the surface and surrounding area to be easily cleaned and disinfected in the event of a spill. While a carpeted floor is not ideal, if you really don't have any other choice, you can make do with a rubber or plastic floor mat under your workstation. 

Though it's nice to have a spacious setup, the Playground only requires about a square meter of physical space, so you should be able to fit it almost anywhere. That said, you'll also want ample room to store safety and cleaning materials, prepare samples, and, of course, for your trusty copy of the Zero-To-Genetic-Engineering Hero book, so if you're not the most organized (this is where some of us start looking around uncomfortably) don't try and put this feature to the test.

The final thing you'll want to keep in mind when choosing a spot is the ambient temperature and air quality. This may seem a bit like overkill, but it's actually one of the most important factors to consider. Too cold a location will make it difficult for your bacteria to grow, so if your basement has even the slightest resemblance to the Arctic, save yourself some trouble and take your Playground upstairs. For the fastidious among you, the ideal temperature range for the experiments we'll be conducting is 17-25 °C (yes, we're doing science now so either keep up, or convert!). Dirty air is also something you want to avoid. Molds don't tend to have the best table manners and so will steal the nutrient-rich media we feed our bacteria right under their (proverbial, of course) noses. 

It may be difficult to tell if your chosen location has any air quality issues before we actually start growing some bacteria  (try to avoid places that are humid or near food!), but if you start seeing some suspicious "stuff" in your Petri dishes, you'll want to investigate this issue further. We'll talk more about what to do if you encounter this problem in the coming chapters, but as a rule of thumb, if you see something growing that isn’t your bacteria, you should inactivate immediately!

 

Some examples of places you can up your genetic engineering workstation include a desk at home, a table at school, or a workbench at your local makerspace.

Some examples of places you can up your genetic engineering workstation include a desk at home, a table at school, or a workbench at your local maker space. 

 

 

So where did I choose to set up my workstation? In a decidedly non-polar basement. For me, it was the ideal spot, since it has plenty of space, as well as close proximity to an empty mini fridge – which is great for storing living material before you're ready to use it – and computer (not required, but you never know when Google – or one of AminoLabs’ virtual simulators – might be helpful!). 

Now, if you own the space you're setting up in then you can just forge on ahead, but for the younger aspiring synthetic biologists among us (myself included) you face the additional challenge of convincing your parents, school administrators, or other supervisors of letting you play with E. coli (cue the screaming) on their turf. My advice (after, of course, explaining to them that this is not the same E. coli that ravaged Chipotle a while back) is that you present them with as foolproof a plan as possible. The more prepared you are, the more likely you are to get their approval. And, hint, hint, if you take the last three or so paragraphs and cut out the occasional snarky remark (which adults don't tend to be the most fond of), then you have the makings of such a plan. It’s also a good idea to give Amino Labs’ Bacteria Safety blog post a quick read if you have any doubts about just how friendly E. coli can be!   

Now, we're almost done, but I did mention something about safety equipment. Make sure that your workstation is equipped with a lab coat and protective footwear and eyewear. Additionally, you should also have some basic cleaning supplies, such as: paper towels, liquid bleach, a spray and/or alcohol cleaner, a trash bin, and a biological waste container. 

What exactly is a biological waste container, you ask? It’s really nothing fancy – a medium-sized metal pail or bucket will do, and it’s just where we’ll place the inactivation bags that come with each kit. While you likely won't need to have all of these items out all of the time, it's always a good idea to keep them relatively close by (or at least know where you can find them) in the event of an emergency.

  I placed my DNA Playground on the countertop in my basement. I make sure to always have paper towels, a spray cleaner, and a biological waste container at the ready (the latter contains my extra cleaning supplies when I’m not experimenting). I have a waste bin situated under the countertop, and gloves, a lab coat, and bleach stored in the overtop cabinet.

I placed my DNA Playground on the countertop in my basement. I make sure to always have paper towels, a spray cleaner, and a biological waste container at the ready (the latter contains my extra cleaning supplies when I’m not experimenting). I have a waste bin situated under the countertop, and gloves, a lab coat, and bleach stored in the overtop cabinet. 

 

 

Yeah, you've probably heard of DNA before, but have you ever seen it? I'm guessing not. Often, we can get so deep into the weeds of biology that it's worth reminding ourselves that the complex processes and machinery we are studying actually DO exist beyond the tidy, colorful visualizations we give them in our textbooks. They're much more messy, chaotic, and alive than the static page can ever really convey. So, on that philosophical note, let's see if we can't squeeze a strawberry hard enough to get some DNA to come out!

As I'm sure you can tell, I'm no fan of the overly pedantic and don't want this to read like a textbook, so I won't be listing out all the materials you'll need to gather before each experiment, or the step-by-step procedure we're going to follow. You'll want to refer to your copy of Zero-To-Genetic-Engineering Hero for those details. 

With that out of the way, we need to explain how we're going to extract DNA from a strawberry's cells. All cells are bound by an outer membrane, and plant cells – like those making up a strawberry – have an additional layer of protection via their cell wall, which is especially sturdy. So the mechanical force of just mashing a strawberry with our bare hands isn't going to suffice here. While pulverizing the strawberry will help disrupt the lattice-like structure that its cells are usually organized in, it will still leave each individual cell mostly intact (have a look at the image below to see what I mean). What we need is some sort of targeted chemical drill that will be able to penetrate through these cells' defenses so that the DNA inside is accessible.

Applying mechanical force to our strawberry (i.e., mashing it up) will only separate the cells from one another, instead of opening them up. So we need to do something extra to release the DNA inside.

Applying mechanical force to our strawberry (i.e., mashing it up) will only separate the cells from one another, instead of opening them up. So we need to do something extra to release the DNA inside. 

 

 

Luckily, we don't have to look far. Common household chemicals like soap, shampoo, and dish detergents all contain these miniature molecular drills, known as surfactants. And they make such good drills because they're what we call amphoteric molecules. This just means that they have both a hydrophilic – or water-loving – part (the head), and a hydrophobic – or water-hating – part (the tail). To accommodate the desires of both, surfactants congregate into wheel-like structures called micelles, in which their hydrophobic tails are clustered on the inside, away from the water (like the spokes of a wheel), while their hydrophilic heads line the outside, towards the water. 

Because a cell membrane is made of similarly amphoteric molecules, surfactants can forcibly integrate them into their own micelles, cutting holes into the cell membrane and exposing the cell's innards. And if we think about it, this makes sense! After all, we use soapy substances to kill bacteria, and the way they do this is by cutting them into (and I mean this literally) a million pieces.

 

(1) The soap molecules called surfactants, attack and cut into the cell membranes of the fruit cells; (2) The surfac􏰎 tants then form spherical jumbles called micelles which contain surfactants and cell debris.

 

(1) The soap molecules called surfactants, attack and cut into the cell membranes of the fruit cells; (2) The surfactants then form spherical jumbles called micelles which contain surfactants and cell debris.

 

 

Ok, so let's do this! Take a plastic bag and pour in about a tablespoon of distilled water. Distilled water doesn't have any charged ions in it, as opposed to the tap water you might get from your kitchen sink. Ions are just like miniature magnets, where ions of the same charge repel while  ions of opposite charges attract. While you can still technically do this experiment with tap water, minimizing the presence of unnecessary chemical species will just make things easier. 

We'll also want to add in about a fourth of a teaspoon of salt. When we break open the strawberry's cells, not only will DNA be released, but also the millions of other molecules that are essential to the cell's proper functioning. We want to isolate the DNA, and prevent it from interacting with any of these other molecules. Since salt is so reactive, it will bind to almost all of these other molecules, creating a "buffer" of sorts around them, and preventing them from interacting with anything else.

Now, add in one strawberry to this solution. Seal the plastic bag and mash to your heart's content. It's actually surprisingly hard work to completely pulverize the strawberry, but if you've done it correctly, you should be left with a relatively smooth fruit slurry without any noticeable lumps. Before we begin to lyse – or break open – the strawberry's cells though, we have to consider how the DNA is already situated inside those cells. And, contrary to the way we often represent it, DNA is not a static molecule. 

DNA is  a superhighway on which little protein "vehicles" can constantly dock, and make local changes to the DNA's shape and state. To remove the proteins that are currently bound to the DNA, we need to employ a clever little molecule called EDTA – or, if you really feel like torturing yourself, ethylenediaminetetraacetate (and, might I point out that I actually had to spell the thing, so I don't want to here any complaints). 

EDTA is very good at binding to positively charged ions – like calcium, magnesium, zinc, and even iron ions – which are what many proteins use to "glue" themselves onto DNA. EDTA will attract these ions more strongly than the proteins they are currently bound to, causing those proteins to disengage from the DNA. In other words, EDTA is a vat of oil we dump all over the DNA expressway, causing any attached vehicles to slip off. This way, when we try to collect the DNA, we'll avoid bringing along any unwanted proteins.

Strawberry DNA extraction step 1 - mash the strawberry

Find some form of soap or shampoo containing EDTA. Since surfactants are the core of any soapy substance, you are guaranteed to already have them inside your soap/shampoo. Add about a fourth of a teaspoon of this substance – known in the lingo as a lysis buffer – to our current mixture, and massage it thoroughly. 

It may seem like nothing has happened, but we've actually liberated DNA from the strawberry's cells. The surfactants contained in our lysis buffer drilled holes into the cell membranes of the strawberry's cells, releasing DNA. EDTA cleaned these molecules up, and the salt we added earlier quickly formed "buffers'' around them, to prevent them from reacting with any other molecules. So why does everything look the same?

Well, even though we've managed to isolate our DNA, it's still dissolved in a solution of salt water, strawberry mush and soap. We need to convert it from its aqueous form (watery liquid) to its solid form, which we can actually see with the naked eye. Before we figure out how to do that though, we’ll want to remove all the extraneous material currently contained in our solution – all the millions of other molecules I mentioned earlier that, at least for the purposes of this experiment, we aren't too concerned about. 

Many of these molecules are quite large, and so we can get rid of them with a simple filtration setup, for example, by passing the mixture through a coffee filter and into a plastic cup. Make sure the filter is well secured (I opted to use a rubber band), as I learned the hard way that the weight of our strawberry slurry is more than enough to cause a precariously balanced filter to cave in on itself. You'll have to be a little patient here, as to get optimal results we’ll need to wait for a few minutes. Don't be dismayed if only a tiny bit of solution remains after the filtration. Even just a half-teaspoon-or-so will yield noticeable amounts of DNA.

Strawberry DNA extraction - filter step

To convert the DNA in this filtered solution from its aqueous to its solid form – a process known as precipitation – all we need to do is fill up a narrow glass tube with isopropyl alcohol (at least 70% concentration, but the closer you can get to 99%, the better your results will be). The isopropyl alcohol molecules are uncharged, unlike the DNA – which has a strong negative charge, due to the phosphate groups making up its backbone – and the water molecules it’s dissolved in – which have weaker, partial charges. 

Currently, the partially positive hydrogens of these water molecules are attracted to the negatively charged DNA, forming a barrier of water molecules around each DNA molecule. When a molecule is encircled in this way by water molecules, it is said to be dissolved, or in its aqueous state. However, if we add a half teaspoon of our filtered solution to the isopropyl alcohol, something strange will happen. A silvery, stringy, substance resembling coiled-up yarn will begin to appear. Ta da, there you go: DNA à la Fraise!

Strawberry DNA extraction result

So, what actually happened? Well, since the isopropyl alcohol molecules were uncharged, they helped break apart the existing attractions between the water and DNA molecules. As a result, some of the DNA molecules were no longer dissolved, and so precipitated – or “fell out” – of the solution. Just like EDTA, you can think of isopropyl alcohol as a lubricant which causes the water molecules to loosen their grip around some DNA molecules, making them visible.

Now, some of you may have done this before. That wasn't really genetic engineering, you’ll say. No, it wasn't – but I promise we'll get to that in the next post. Simple as it may be, I think it's worth pondering just how amazing what we did is. We just extracted all the biological code needed to create a multicellular organism as complex as a strawberry! 

We were able to take this abstract biological concept that all of us know about – DNA – and see what it actually looks like in real life. And the fact that that this slimy and gross goo – something that we all would have probably thrown out, if we didn’t know what it was – has these amazing emergent properties that are in no way obvious or expected, and is at the same time responsible for you, me, and every other living being on this planet existing, makes the whole thing all the more beautiful.

So, if nothing else, let this experiment be an instruction in humility. Though genetic engineers are capable of some pretty amazing feats, even we are still humble wielders of tools far more complex than any human creation, tools we still don’t completely understand, tools like DNA. At the same time, be inspired! There is this amazing microscopic world that so many of us completely ignore in our day-to-day lives, and today we got a glimpse of what it looks like. 

But I’m going to go out on a limb here and guess that you probably aren’t content with just a glimpse. Neither am I, and I promise we’ll be back for more. Truly, DNA extraction is just the first step. Next time we'll be channeling our inner Picasso as we learn the basics of culturing bacteria and brave the avant-garde world of Bio art!