Dna Barcoding

DNA Barcoding
Click on the following link and view the background on DNA barcoding:
https://www.dnalc.org/resources/animations/dna-barcoding.html
1. What is a DNA barcode?
DNA barcoding is a fast accurate method of identifying plants and animals, or products made from them. DNA barcode is DNA sequence that uniquely identifies each species of living things. Short DNA sequences are used to identify species by comparing them with the known barcodes in large databases.

When you get to the section where the animation separates into plant and animal cells, select animal cells: 2. What are the two steps taken at the beginning of the process to break down the cell membrane? a. In order to dissolve organelles lysis solution is added. b. A pestle
is used to crush up the tissue. This allows tough components of cells to be broken down such as cell walls.
3. What is the first step to separate the cellular debris from the DNA?
The first step to separate the cellular debris from the DNA is centrifugation. This allows the cellular debris to be put at the bottom of the tube in order to be collected.
4. After spinning the sample-do we keep the pellet or the supernatant?
After spinning the sample we keep the supernatant.
5. What is the purpose of adding the silica?
Silica binds to nucleic acid when there is lysis solution.
6. What is the purpose of washing the silica pellet?
Washing buffer gets rid of the impurities in the sample. Since silica is insoluble in the buffer the silica can decompose or remain as a pellet.
7. Why do we add water to the pellet in the final step?
To elute the DNA off the pellet.

PCR (Polymerase Chain Reaction)
Click on the following link and perform the PCR experiment:
http://learn.genetics.utah.edu/content/labs/pcr/
1. What is the purpose of PCR?
Create billions of copies of a target DNA sequence. These copies are identical.
2. What is special about PCR tubes?
PCR tubes are perfect for heat transfer. Evaporation is averted when tubes placed in thermal cycler.
3. What is the function of primers?
Primers bind to the target segment at either end. They also replicate DNA sequence. Primers are so specific they only have a small change they will target the wrong sites.
4. Explain why you add nucleotides.
Nucleotides generate tons of DNA copies.
5.
What is the function of DNA polymerase?
DNA polymerase generate DNA copies by inspection the DNA code and binding to nucleotides that match.
6. What is special about the DNA polymerase used in PCR?
DNA polymerase in PCR is able to handle high heat.
7. Name the apparatus that will heat and cool the sample.
DNA Thermal Cycler
8. Why is the sample heated to almost the boiling point?
At this temperature the double strands of DNA are pulled apart into two single strands.
9. Why is the sample then cooled?
This temperature the single strands can rejoin before this occurs primers attach to their target sequence.
10. Why is the sample then brought up to 72 degrees Celsius?
DNA polymerase is turned on at this temperature allowing it to add the corresponding nucleotides on a DNA strand by locating a primer attached to a DNA strand.
DNA Gel Electrophoresis
Click on the following link and perform the gel electrophoresis experiment: http://learn.genetics.utah.edu/content/labs/gel/ 1. What is the function of gel electrophoresis?
Way to sort and measure DNA strands according to length. This technique is also useful for separating other types of molecules, like proteins.
2. Where are DNA samples placed in the
gel?
DNA samples are placed into wells at one end of the gel.
3. What makes the DNA move across the gel?
By adding electrical current DNA moves.
4. Why do DNA fragments of different lengths travel to different locations on the gel?
Short fragments move through the wells in the gel more quickly. Over time shorter fragments in the sample will move further away from the starting point than the longer fragments.
5. Why is the DNA stained?
To make the DNA visible to the naked eye.
6. Is each band on the gel made up of only one DNA segment?
Each band contains DNA molecules of one size fragments.
7. Explain the function of each of the following pieces of lab equipment. a. agarose- Used as diagnostic tool to visualize the fragments b. gel mold- Allow gel to presume a shape c. gel comb- Create wells in gel in which sample is inserted d. buffer- Used to provide ions that carry a current to maintain the pH at a relatively constant value. e. microwave- Allows for the melting of agarose in TBE f.
electrophoresis box- Allows voltage to be applied to the gel g. DNA size standard- Set of standards that are used to identify the approximate size of molecule run.
8. Does DNA have a positive or negative charge?
DNA has an overall negative charge because of the phosphate groups in the DNA backbone.
9. Which electrode does the DNA move toward?
Toward the positive electrode.
10. What indicates that the electric current is running?
The movement of molecules from one end of the gel to the other.
11. Name the chemical used to stain the DNA?
We used Sybrsafe to stain the DNA but ethidium bromide can also be used.
12. What is used to reveal the stained DNA?
By putting the gel in ultraviolet transilluminator
13. What two things in this experiment can be dangerous and why? a. Electrophoresis units at 100 volts can provide a lethal shock b. Acrylamide has hazards it’s a nerve toxin/ ethidium bromide is carcinogenic.
14. What protective equipment can be used to minimize the danger? a. Wear gloves b. Using power supplies with safety features that detect issues with electrical circuit
15. What were the estimated lengths of your DNA fragments?
a) 6000 bp
b) 3500 bp
c) 1500
bp