Lab 11 DNA Technology

LAB 11 

DNA Technology

Part 1. DNA Extraction

This exercise was the removal of DNA from a cheek sample. This process can be used for genetic testing,body identification, and analysis of forensic evidence.

1. Swab the inside of your subjects cheek. Cheeks shed thousands of cells a day. 

2. Cut the end of the swab off into a test tube with a lid and add lysis solution. Lysis means to separate. Then add the tube to warm water.

3. Remove liquid from water then add concentrated salt solution with a micropipettor. This causes cells and proteins to clump together.

4.Place the sample into a centrifuge with a counter balance water sample and spin the sample. This action will cause the proteins and cell debris to sink to the bottom of the test tube.

5. Remove top liquid with micropipettor and add isopropyl alcohol. Invert the tube several times to mix alcohol. Since DNA is not water soluble the DNA will clump together.

6. Spin the sample again- remove liquid and allow DNA to dry. This DNA sample can now be mixed with any solution and frozen for many years. 

Question: What specific type of tissue is that inside the mouth? 

Answer: The specific type of tissue is called: "Stratified Squamous" this tissue is thick and has multiple layers to provide protection. It's found lining body cavities like the mouth and outer layer of the skin.

 Part 2. Electrophoresis 

Gel Electrophoresis is a process that separates DNA stands according to lengths. The Gel is a Jell-o like material that has tiny holes that allow strands of DNA to move through it with the addition of electric current.

1. The gel needs to be made using powdered agarose, buffer,flask,microwave,and a gel mold and gel comb. The end product will be a gel mold that has empty wells on one end of the mold to allow for DNA samples. The gel mold will then be placed into a electrophoresis box with another buffer. The buffer will conduct a electrical current from one end of the gel to the next. Now the DNA sample is ready to be placed in the gel.

2. There will be three different samples to be loaded into the gel. Loading buffer, DNA sample to separated into lengths , and DNA standard size which contains long DNA stands of determined length. The unknown DNA sample with be compared later to the standard size to determine length of unknown DNA sample.

3. The DNA sample is sucked up with a clean micropipettor tip and placed into the first empty well in the Gel mold.

4. Anew clean pipet tip is used to suck up the DNA standard sample and placed into the second empty well in the Gel mold. This will be used to compare DNA strand lengths.

5. It's now time to add electricity to the Gel. The Gel mold is placed into a Gel box and the negative charge wire (black) is connected to the black post and the positive charge wire (red) is placed on the red post of the power supply box. Together an electrical current is ran through the gel. Since DNA has an negative charge the gel mold with the wells will be placed at the same end the negative (black) wire is connected. This will allow the DNA to move through the gel.

6. The electricity is introduced to the gel and electrophoresis is in progress. Tiny bubbles on both ends of the gel will indicate that current is moving through the gel.

7. This process is where the DNA strands are separated. This will explain why DNA fragments separate out in bands in the gel. As mentioned DNA have a negative charge which means they will move towards the positive charge on the other end of the gel mold. Again, this mold have tiny holes all through it to allow DNA movement. Short DNA stands will move through the gel faster than the long strands. Over time the short strands will move further away from their starting point. we can't see this process with the short strands but with the added buffer we can see the long strands as they move.

8. At this point the gel mold is removed from the electrophoresis box. The samples are ready to be analyzed and the DNA strand lengths can be determined.

9. The DNA now needs to be stained using ethidium bromide which binds to DNA and provides color that will show under a florescent light. Under this light we will be able to see large groups of DNA strands that show up as bands in the gel.

10. Lastly, the gel mold is moved out of the staining solution and placed over a Ultra-UV light box.

Now the lengths of the unknown DNA sample can be determined by comparing the results to the standard DNA sample with the determined lengths.

These were the results

 

 These were my predictions

 My predictions were correct.

 

       An application for electrophoresis can be used for many things. DNA analysis, Protein analysis, antibiotics analysis, and vaccine analysis.  Although I liked the application for vaccine electrophoresis the best because it gave great examples of vaccines that have been created by electrophoresis like the FLU, hepatitis, and polio the process cannot be provided due to confidentiality reasons so I would be done!! 

     Antibiotics electrophoresis is the next thing I liked. I found a process that uses paper electrophoresis to find new antibiotics. This has been used for some time in laboratories. This technique involves putting a culture on a piece of paper and then soaked in a buffer. The strips are tested by electrophoresis by the technique of Kunkel and Tiselius: "A simplified procedure for filter paper electrophoresis is described in which disturbing factors such as evaporation, heating, buffer concentration gradients, and pH changes in the electrode vessels were reduced to a minimum." In this instance the antibiotic chloromycetin was used. Chloromycetin was placed on the paper and on both sides of the culture and after migration the position of this antibiotic was reveled. This process helps in determining if the antibiotic is acidic, basic, or amphoteric to the culture.

References

"Applications of Electrophoresis in Studying New Antibiotics." 1953 Nature Publishing Group, 16 May 1953. Web. 15 Nov. 2015. 

 

 

"ELECTROPHORESIS OF PROTEINS ON FILTER PAPER." ELECTROPHORESIS OF PROTEINS ON FILTER PAPER. Web. 15 Nov. 2015.