DNA Extraction
In order to determine an individual’s microsatellite genotype or its life history stage, we have to first extract it’s DNA.
DNA, or deoxyribonucleic acid, is the molecule that encodes the genetic instructions used in development and functioning in living organisms. It is composed of four different types of nucleotides.
There are three types of DNA that we work with in the Sotka lab:
DNA, or deoxyribonucleic acid, is the molecule that encodes the genetic instructions used in development and functioning in living organisms. It is composed of four different types of nucleotides.
There are three types of DNA that we work with in the Sotka lab:
- nuclear DNA
- DNA from the chloroplast
- mitochondrial DNA
Nuclear dna |
DNA from the chloroplast |
 Nucleus
The first is nuclear DNA from the nucleus of a eukaryotic cell that houses genetic information. We use certain markers found in this type of DNA in all the organisms we study, animals, plants and seaweeds.
mitochondrial dna Mitochondria
Finally, we work with mitochondrial DNA, which is found in the mitochondria, or the cellular powerhouses because they generate the cell’s energy in chemical form, in the animals, plants and seaweeds we study.
|
 Chloroplast
The second is the DNA from the chloroplast, a specialized organelle in algae and plants, where photosynthesis occurs.
Learn more about chloroplasts and photosynthesis here! |
dna extraction technique
DNA Extraction Technique
First, we have to mechanically open up the cells by homogenizing the plant or animal material. Homogenize simply means to make uniform or make similar.
For animals, we take preserved tissue that has been stored in ethanol. The ethanol fixes the tissue so the DNA does not degrade. The type of tissue depends on the organism. Figuring out which type of tissue requires a lot of trial and error. Researchers use tissue from hair to fin clips.
For all seaweeds, including Gracilaria vermiculophylla, we dry the algal material with silica gel. This is what is called a desiccant and removes water from the algal pieces and dries them, preserving the DNA like ethanol does for animal tissue. What we use is very similar to the little packets found in new purses or sometimes in a new shoebox. Once the material is dried, we use our mixer mill to grind the material into a very fine powder.
We then added what is called lysis buffer to the ground seaweed tissue or to the animal tissue. The lysis buffer causes the cells to open.
For animals, we heat the tissue and lysis buffer mix to 65 degrees Celsius (that’s about 150 degrees Fahrenheit). But, during her PhD, Stacy, and the lab she worked in which she performed her dissertation research, found room temperature worked better for seaweeds rather than heating the mix to 65 degrees Celsius.
The resulting mix is called a lysate. But, it’s not just DNA, but other things, such as cellular debris (i.e., trash) or polysaccarhides (i.e., sugars).
We remove the gunk by centrifuging. This step spins our tubes or plates really, really fast. This spinning forces all the gunk to the bottom of the tube.
We, next, pipet the cleared lysate to a new tube or a new plate. The binding buffer creates conditions that are optimal to bind the DNA, which has been floating in the lysate mix, to a silica membrane.
We pass the lysate and binding buffer through the silica membrane by centrifugation or by vacuum pressure (see Vacuum manifold page). This step pulls the excess lysate mix through the membrane into a waster container. The DNA, meanwhile, has become bound to the membrane.
After the DNA is bound, we wash the DNA with two different types of buffers that are much like a detergent you wash your clothes with. Laundry detergent washes the dirt off your clothes. Our wash buffers, wash the remaining gunk off our DNA.
Finally, we elute the DNA. Elution is a process of extracting the DNA off the silica membrane by washing with really pure water or with a buffer provided in the kit. The wash buffers and elution steps require the use of the centrifuge or the vacuum manifold.
Then, we have our DNA ready to go! See the equipment and techniques page for more information on the equipment used for the extraction or what we will use the DNA for later on at the lab bench!
First, we have to mechanically open up the cells by homogenizing the plant or animal material. Homogenize simply means to make uniform or make similar.
For animals, we take preserved tissue that has been stored in ethanol. The ethanol fixes the tissue so the DNA does not degrade. The type of tissue depends on the organism. Figuring out which type of tissue requires a lot of trial and error. Researchers use tissue from hair to fin clips.
For all seaweeds, including Gracilaria vermiculophylla, we dry the algal material with silica gel. This is what is called a desiccant and removes water from the algal pieces and dries them, preserving the DNA like ethanol does for animal tissue. What we use is very similar to the little packets found in new purses or sometimes in a new shoebox. Once the material is dried, we use our mixer mill to grind the material into a very fine powder.
We then added what is called lysis buffer to the ground seaweed tissue or to the animal tissue. The lysis buffer causes the cells to open.
For animals, we heat the tissue and lysis buffer mix to 65 degrees Celsius (that’s about 150 degrees Fahrenheit). But, during her PhD, Stacy, and the lab she worked in which she performed her dissertation research, found room temperature worked better for seaweeds rather than heating the mix to 65 degrees Celsius.
The resulting mix is called a lysate. But, it’s not just DNA, but other things, such as cellular debris (i.e., trash) or polysaccarhides (i.e., sugars).
We remove the gunk by centrifuging. This step spins our tubes or plates really, really fast. This spinning forces all the gunk to the bottom of the tube.
We, next, pipet the cleared lysate to a new tube or a new plate. The binding buffer creates conditions that are optimal to bind the DNA, which has been floating in the lysate mix, to a silica membrane.
We pass the lysate and binding buffer through the silica membrane by centrifugation or by vacuum pressure (see Vacuum manifold page). This step pulls the excess lysate mix through the membrane into a waster container. The DNA, meanwhile, has become bound to the membrane.
After the DNA is bound, we wash the DNA with two different types of buffers that are much like a detergent you wash your clothes with. Laundry detergent washes the dirt off your clothes. Our wash buffers, wash the remaining gunk off our DNA.
Finally, we elute the DNA. Elution is a process of extracting the DNA off the silica membrane by washing with really pure water or with a buffer provided in the kit. The wash buffers and elution steps require the use of the centrifuge or the vacuum manifold.
Then, we have our DNA ready to go! See the equipment and techniques page for more information on the equipment used for the extraction or what we will use the DNA for later on at the lab bench!
