A Sweet Way to Learn Molecular Biology

On Saturday March 29th Mariola Kulawiec and Lawrence Own led HiveBio’s first Electrophoresis class. Four eager students learned about gel electrophoresis using the artificial colors in candies as a bright (and sticky) example of how this technique can be used in chemistry and biology.

Lawrence with gel tray

Lawrence kicked things off with a brief presentation on the theory behind gel electrophoresis and how it’s used to test properties of DNA, RNA and proteins in biological experiments. He then explained to participants what they will be doing in the lab. Step one: use isopropyl alcohol (rubbing alcohol) to extract the coloring agents from various candies. Step two: Separate the colors using gel electrophoresis!

Experimental setup

Class in sessionOur students first had to design their experiment. There were many candies to choose from, but only room on the gel for each person to test a select number of samples. How to choose? Compare single colors across candy types? Make a rainbow? Go for your favorite flavors?

As students set up their candies for extraction, Mariola went over how to keep a record of their experiment using a handout, and emphasized the importance of labeling. In fact, everybody in the room who had worked in a lab before agreed on the importance of having everything labeled clearly. Lawrence explained how to make an agarose gel, and supervised while students prepared one of their own.

Making the gelDuring preparation, the candies were soaking in alcohol, but it seemed like the extraction was going very slowly, if at all. What could be the problem? Were the candies used not very well suited to the experiment? Did they need more mixing? Was there a difference between shaking the sample cups and using a pipette to run the alcohol over the candies? What about confectioner’s wax; could the candies be coated? What would happen if the M&Ms were crushed; would the alcohol extract colored compounds from the chocolate?
As often happens in science, one experiment sparked the ideas for many others.

After a brief adventure with a leaky gel tray, students broke out the micro-pipettes and began making standards from commercial food coloring. Working with tiny volumes of food coloring was good practice for handling the extractions.

Loading the gel 1

Loading the gel for electrophoresis requires a steady hand.

And then, it was time for the big challenge – loading the gel! There was one sample too many, and a brief paper-rock-scissors competition ensued to see who would leave out one of their food coloring standards. Loading the gel correctly was a test of lab notebook record-keeping, steady hands, spatial awareness, and tube labeling. During the process a few of the small tubes fell through the rack, but thanks to accurate labeling, no samples were lost!

Finished gel

After some waiting, chatting, and ducking out of the lab to eat some of the untested candies, the gel was done!

Despite challenges and set-backs, a good time was had by all. The class generated some great discussion about results, future experiments and troubleshooting experimental methods for the next round of Candy Electrophoresis! Join us next time!

Veenadhari explains 1

Veenadhari Kollipara explains gel electrophoresis to friends and family

Mariola and Lawrence both work for Invention Evaluator.
Mariola is also the founder of Witty Scientists (facebook.com/WittyScientists).

Photos and Correspondence by Christine Lloyd

DRD4 Project Update, and the Basics of Gel Electrophoresis

The Dopamine Receptor D4 (DRD4) gene contains a segment 48 base pairs long, which repeats between 2 and 11 times, depending on the individual. This pattern in DNA is referred to as a VNTR (Variable Number Tandem Repeat). Studies have shown particular phenotypes, or traits, in people who have exactly 7 repeats of this DRD4 VNTR. Such people may exhibit characteristics such as a novelty seeking personality, susceptibility to ADHD, longevity, a nomadic lifestyle, and other traits.

Michal, Zeb, Noah and Georgia have been developing and troubleshooting a method in the HiveBio lab to determine the number of these repeats in the DRD4 gene of our research participants. They begin by taking a buccal (cheek) swab to gather cells, and then make various dilutions of the cell sample in a chemical buffer that exposes the DNA.

20140208_172411

Developing the method for isolating DNA

Next they amplify the number of DNA strands in their sample by using a technique called Polymerase Chain Reaction (PCR). The amount of DNA containing the DRD4 gene is so tiny compared to the total amount of DNA in our cell sample, that we need to make more copies of this part of the DNA in order to analyze it. In order to amplify our DNA, we add it to a molecular cocktail of all the ingredients needed to make more copies of the DNA segment of interest. This cocktail includes very specific molecules called “primers” that specify the region of the DNA to be amplified. Design of the primers is one of the first and most important steps for a research study of this nature. Once the DNA of interest is copied over billions of times using a machine called a thermocycler, we have enough to separate it out in a gel.

Noah, Georgia and Zeb set up the gel

Noah, Georgia and Zeb set up the gel for electrophoresis

HiveBio's first electrophoresis gel!

HiveBio’s first electrophoresis Gel, February 2014

Some might just see a bunch of tick marks on a clear gelatin-like substance, but to those savvy in molecular biology techniques, this “gel” shows the relative sizes of the DNA being examined. The DNA is stained with a blue dye so we can see how far it has run down the length of the gel. The gel is made in the same way you make Jell-O at home. You heat a powder, in this case agarose, in a certain volume of water, and then allow it to cool in a rectangular mold. In this case, the gel is made from a 2% solution, so 2 grams of agarose powder for every 100mL of water. We put a comb at the top so when the gel solidifies we have wells in which to load our DNA and keep our samples separate.

When solidified, the agarose produces a homogeneous gel matrix through which DNA can diffuse when an electrical gradient is applied. The side of the gel where we load the DNA is the negative end, and the far side is the positive end. DNA has an overall negative charge, and its rate of diffusion through the gel is dependent on the length of the DNA fragment, also referred to as its size. By looking at the relative distance the bands have traveled, we can estimate the size of the DNA fragments we’ve loaded. The smaller the pieces, the farther they will go in a shorter amount of time! Once the DNA had moved most of the way down the gel, as indicated by the blue loading dye, the team stopped the gel electrophoresis and took photos for analysis.

GelGreen

Lanes: 1. Ctrl Primer A 2. PrimerSet1, Participant 1, 3. PrimerSet2 Participant 1, 4. Neg Ctrl, 5. Ctrl Primer B 6. PrimerSet1 Participant 2 7. PrimerSet2 Participant 2 8. DNA ladder

Lanes 1 and 5 are the positive controls, samples of DNA amplified using the control primers. Lane 4 is the negative control, no DNA. Lanes 2 and 3 are DNA samples from participant 1, using primer set 1 and primer set 2 respectively. Lanes 6 and 7 are DNA samples from participant 2, using primer set 1 and primer set 2 respectively. Lane 8 is the DNA ladder, a standard set of DNA lengths.

Ideally, we should be able to determine the number of VNTRs by looking at the length of DNA in our gel as compared to the standard sizes in the ladder. However, not everything always goes according to plan! The wavy lines of the DNA ladder indicate that something is impeding its diffusion through the gel. The team will have to troubleshoot this to get a clearer and more accurate picture.

So how many DRD4 repeats do our two participants have? Stay tuned to find out…

If you are interested in learning more about the DRD4 Project at HiveBio or have questions about developing your own project to work on in the lab, please contact Zeb Haradon or Michal Galdzicki at hivebio@gmail.com

The Thermocycler used for this project is a GeneAmp9600 generously donated to HiveBio by Rob Carlson.