Methods, Methods, Methods (My Research, Vol. 5)

_{By geralt on pixabay.com}

On Tuesday, I can finally take the next big step in my project for my Bachelor thesis. Because of this, I wanted to show you some of the results I got so far, the long way you have to go before you can even do something that might lead to interesting results.

And while I was cropping pictures, adjusting colors and reducing image size, I realized one thing: Those of you, who don’t have a background in biology (aka: The majority of my readers) will have no idea what the hell I am even talking about.

Therefore, I decided to make a different post first.

This is said post. And it’s about methods. Methods I’ve been using, some of them are used on a daily basis in biology labs all around the world. I’ll try to make it as easy as possible (and thus not necessarily super-scientific), those who have already worked in a lab might want to skip this.

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Polymerase Chain Reaction - The PCR

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You have a piece of DNA. You want more pieces of DNA. Sadly, you don’t have any samples left to extract it from. That sucks. What do you do now?

Luckily, the replication of DNA is something all living things do all the time. There are certain enzymes that are specialized in making a lot of DNA out of a little bit of DNA. Scientists were able to isolate those enzymes and create a way to easily multiply DNA, without needing a living organism for that. The process is called PCR._@suesa

What happens during a PCR?

_{By Enzoklop (Own work) [CC BY-SA 3.0]}

Thanks, Wikipedia. That was easy.

Okay, maybe not.

So, you have this double-stranded DNA (need a reminder what DNA is? Click here). You then heat it (= denaturation), so it splits into two strands. Tiny pieces of single-stranded DNA (= Primers) attach to these longer single strands (= annealing). Then, an enzyme called polymerase starts synthesizing a new strand of DNA, matching the one that already exists (= elongation). When that is done, you have two double strands instead of the initial one.

And then you repeat. Over and over and over again.

This gets you a lot of DNA, pretty fast. Neat, isn’t it?

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Gel Electrophoresis

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The only method I’m using more frequently than the PCR is gel electrophoresis. It’s good for checking the size of a DNA fragment or sorting DNA fragments after size.

For that, you first need a gel. The gels usually consist of agarose (a sugar) and some kind of buffer. Both are mixed, heated, and then poured in a form to let cool. There’s also a small comb inside the form, which creates tiny “pockets” inside the gel. That’s where your samples are put into.

When the gel is hard (okay, it’s usually kind of wobbly), it’s put in a chamber which is filled with the same buffer the gel was made with. Then, you put your samples into the pockets. And then … you turn on the electricity!

DNA is charged slightly negative, which will make it move towards positive charges. This is used to make the DNA “wander” through the gel, as at the upper end, there’s a negative charge and at the lower end a positive one. Bit by bit, the DNA wanders through the gel. Bigger fragments are a lot slower than smaller fragments, which allows a separation by size.

_{A gel chamber with gel, buffer, and samples. Picture was taken by me.}

You just need to keep an eye on the whole thing (has to run about an hour, usually), as you don’t want to lose small fragments. They will just run out of the gel if you wait for too long.

When the gel is finished, you drench it in ethidium bromide or something similar toxic. That stuff binds to DNA and is visible under UV light. That looks a bit like this:

_{Also taken by me. Colors have been inverted for better visibility, originally the bands were glowing and the background was black.}

With the help of this, you can now determine several things: Did my PCR work? Did my digestion (= cutting DNA into specific pieces by using enzymes) work? Did process that produces a certain kind of DNA work?

You can even cut out pieces of the gel and extract the DNA!

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Transformation, Cloning and Plasmid isolation

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Sometimes, a PCR is not the ideal way to create the product you want. Sometimes, actually, pretty often, you need to fall back on bacteria. And for that, you need plasmids.

What is a plasmid?

Generally, a round piece of DNA. Bacteria can take in these pieces and use them to, for example, become resistant to antibiotics if the DNA is coding for this specific ability. And as with most useful tricks from nature, scientists found a way to adapt this too.

It’s relatively easy to create a plasmid with the DNA you want. In addition to it, you need some other things I’m not going to mention because eh, you won’t remember anyway. The only thing I AM mentioning is the marker. The marker is usually a gene that is “visible” in a way, as the gene we want to insert doesn’t always have a visible effect on the bacteria that contain it.

Commonly used is a gene that provides resistance to a very common antibiotic.

I know, I know. We don’t want antibiotic resistance. That’s exactly the reason why it’s important to handle these kinds of organisms with a lot of care.

Anyway, the bacteria without plasmid are combined with the plasmid and then forced to take up this artificial DNA (mostly either by using a heat shock or by technically electrocuting them). They are then left to grow on, for example, a plate of medium that contains the antibiotic mentioned before. Those that contain the plasmid survive. Those that don’t … don’t.

The surviving bacteria are then put into liquid medium, so they can grow a lot. Then, they’re taken out of their broth, centrifuged a lot and broken open, to isolate the plasmids. Because while the cells were growing and replicating, they replicated the plasmid too and now we have A SHITTON of the plasmid.

And then? We can use the plasmid for stuff. Like a PCR.

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In vitro transcription - IVT

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Last one! If you’ve read the post about the building blocks of life I linked earlier, you should know that there is not only DNA but also RNA.

In biology, the usual way (it can happen differently too) is:

DNA -> RNA -> Protein

The step between DNA and RNA is called transcription, as the information on the DNA is transcribed into RNA to be translated into proteins.

Easy peasy.

In vitro transcription is exactly that, just inside a test tube instead of a cell.

That …. That’s it. You just combine a bunch of colorless liquids, put it in a warm place, wait for two hours and then put a tiny bit of it on a gel for gel electrophoresis to see if it worked.

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If you still have questions (you probably do, this was just a really rough summary), feel free to ask in the comments. I will soon post my results so far, but they will only make sense if you’ve at least somewhat understood the methods.

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Read some more about this:

In Vitro Transcription

Gel Electrophoresis

PCR

Plasmids

DNA Cloning

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Previously:
All about the Sperm-DNA - Vol. 1

Gene Editing - Cre/loxp - Vol. 2

Let's Splice! - Vol. 3

Lab Mouse Care - Vol. 4

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_{GIF was created for me by @saywha and @atopy , rest of the signature by @overkillcoin}