Extractions, Amplifications, and Gels; Oh My! | #2

The first month of me working in the lab consisted of me running DNA extractions twice a day. I would come into work at 8:30 every morning and go down into the basement, past the gross anatomy lab (that is the title on the door not a descriptive adjective, although the smell of formaldehyde is not in my top 10 favorite odors), and grab a block of dry ice to put in the Styrofoam cooler that would hold the hyena samples for the day. Before I could work on human samples, my boss wanted me to get hands on lab work with a high biomass sample. This is because when a low biomass sample (like amniotic fluid or placenta swabs) gets contaminated, the contamination accounts for a greater amount of the microbial fingerprint since there weren’t many microbes to begin with. However, when we are working with things like feces for example, the amount of microbes present goes up drastically so contamination will account for less of the microbial presence. It is because of the effect of biomass on contamination that my boss wanted me to get a lot of practice treating the hyena fecal samples as if they were low biomass samples, that way when I finished NIH training to work with human samples, I would feel comfortable and confident knowing I wasn’t ruining all the science we were doing with the bacteria present on my body.

 

If you want to go into detail about how extractions are done, feel free to look up “Qiagen PowerLyzer DNA Extraction Kit user protocol.” But basically, what I would do is take a 0.25g of hyena poop and put it in a bead tube that contains little beads and a solution. We put it in a machine called a bead beater to lyse open the cells and get the proteins and DNA out in the solution so we can access it. To separate the DNA from the proteins and other cellular components, we centrifuge them which separates the solution into a pellet at the bottom (the stuff you don’t want) and a supernatant (a solution with the DNA in it). After a series of centrifugations, we clean the DNA with an alcohol and then release it from the spin filters membrane using a more polar solvent. In the end, we have approximately 50 microliters of DNA.

One of the DNA extractions kits used for hyena samples. Fun fact: our lab is very thorough and nervous about contamination so in order to keep any contamination constant not only did we call the company and make sure that each kit was from the same lot, but each solution inside the kit has to have the same lot number. That means that each of the kits we bought was made at the same time by the same people to ensure consistency in our contamination when/if any is found.
This is the bead beater with the lid up so you can see inside. Behind that black circle are slots to put tubes and when you cap it off tight and close the lid, you can start the machine. It scared me the first time because it sounds like a jackhammer is in the room.

To make sure that we have obtained bacterial DNA, we first need to amplify the DNA that we extracted. First, we take a portion of the extracted DNA and use a technique called Polymerase Chain Reaction, or PCR for short, to amplify the amount of DNA in a small subsample. This happens by combining the DNA, primers for the segment of DNA that we are trying to detect, and a mixture that contains the necessary enzymes for DNA replication and a dye for later visualization. After the mixture is made for each sample, it is put in a thermocycler that cycles through different temperatures that help denature the DNA, anneal the primers, and “zip up” the DNA. This happen 32 times, each time with the DNA multiplying exponentially.

This is the thermocycler. When you open the lid up, the machine has little holes to put tubes in that run in rows. These rows have heating strips beneath them so they can cycle through temperatures.

After we have our PCR product, we want to be able to visualize that our amplification worked. To do this, we run an electrophoresis gel. If you haven’t taken a science class (or you don’t remember because let’s be real, you take a lot of classes and sometimes information slips out of your brain), electrophoresis capitalizes on DNA’s negative charge and different fragment sizes. Whenever I do PCR, I follow it up by making a gel and running it. I load up the wells with the PCR products and fill the first well of each row with a DNA ladder that shows multiple bands that correspond to different length DNA fragments. The ladder will allow me to see that our bands from our PCR products correspond with the known fragment length of the section of DNA we are trying to multiply. Once each product is loaded into a well, we run the gel at 85V for around 30 minutes, remembering to run it from negative to positive so that the DNA travels down the gel and not off into the buffer surrounding the gel.

These are electrophoresis units (gel boxes), one big and one small. I usually only use the small one with 0.5 x TAE buffer since I rarely need more than 20 wells. There are no gels in here currently or I would get a picture of the wells, but I don’t want to use materials when we don’t need a gel for anything. Sorry!

After running the gel, we visualize it by taking a picture of it under UV light. Under UV light it comes out quite orange but after capturing the picture, we Photoshop the image so that we finish with an inverted black and white photo.

This is a photoshopped gel image. On the left you can see the ladder. Notice how our bands to the right of the ladder match up with one of the bands on the ladder indicating that we do have bacterial DNA. The difference in saturation is due to differences in concentration of DNA in solution. The brighter the band, the more nanograms per microliter.

This is by no means the last step in obtaining the sequence for a DNA sample, but after this point, the rest of the lab work is sent off to a lab in New Jersey or to my favorite place on earth aka Ann Arbor to run Sanger sequencing. Once we get the sequence of nucleotides back, the work is mostly on the computer running a program called Mothur developed by a U of M genius (Pat Schloss). I’m not very involved with what happens once we get the sequences back since I’m not very tech savvy, however, I have learned a lot about the program and have gotten the privilege to run through some old data and fool around with it a little bit.

 

Even though what I just described here was for hyena samples, the human samples go through the same type of process just with a different or modified kit since they are considered low biomass samples like I discussed in the first paragraph. This whole process takes around 8 hours when I am running only one set of extractions that has 12 samples (the extraction alone take the entire morning). But depending on the project, we will often do all the extractions first, running a PCR only when we open a new kit or finish a kit. This means we will run two sets a day, one before lunch and one right after.

 

I hope you enjoyed learning about a typical day in the lab for me in the first month as much as I enjoyed living it!

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