Part 1
Here we will be performing a DNA Extraction.
Let's begin this technology assignment with visiting the virtual lab site:
As with any laboratory experiment, we must begin with an acquisition of materials and equipment for it. As I collect the supply, I will be explaining how we will be using it and for what.
1) Buccal swab
Collection of the specimen
2) Eppendorf tubes, three count
Mixing, viewing, and storing the specimen
3) Scissors
Cutting off the top of the swab
4) Micropipettor
Extracting, transporting, and expelling droplets of the liquid with the specimen
5) Lysis solution
This is a unique mix of kind of a detergent and enzyme K. (The DNA wrapped around special proteins called histones, and the K enzyme releases them and unwraps the DNA)
6) Warm water bath
Not this one.
This one!
7) Concentrated salt solution
Causing the proteins and other debris clumps together
8) Micro-Centrifuge
Spinning apparatus. Separation of clumped waste from the rest of liquid in the Eppendorf's tube, here we need an extra tube with just water in it to balance the centrifuge
9) Isopropyl alcohol
We can see the undissolved, clumped DNA
Q: What is the specific type of tissue inside the mouth?
Part 2
Electrophoresis
Electrophoresis is a technique used in laboratories to separate macromolecules based on size. The method applies a negative charge, so proteins move towards a positive charge. This is used for both DNA and RNA analysis.
And now we can go to another site and perform this lab:
And now we can go to another site and perform this lab:
EQUIPMENT AND MATERIALS NEEDED
AGAR GEL (Background - Agar is a gelatinous substance found in the cell walls of certain red marine algae, particularly those in the genus Gelidium. It is used to make vitamin and drug capsules, as a dental impression material, as a base for cosmetics, and as a culture medium for bacteria and other microorganisms. In foods, it is used as a moisture retention (anti-drying) agent in bakery products, in the preparation of rapid settling jellies and desserts, and as a temporary preservative for meat and fish in tropical regions. While a very expensive, highly purified form of sugar, called agarose, is needed for separating DNA or proteins in gels, several kinds of agar can be used for separating scientific stains in gels. These less expensive agars include agars used for microbiological culture media and agar available from health food stores and Asian markets. (Note: Unflavored gelatin, such as Knox“, does not work. The gelatin has a greater affinity to surfaces than to itself, causing the wells to tear out of the gel when the comb is removed. Also, gelatin melts at a much lower temperature and a gel prepared from gelatin is likely to melt while running.)
Gel form and comb
Electrophoresis chamber,
Electrophoresis power supply
Salt solution
Scientific stains to use as samples
Sample-loading device
Masking tape, if needed to seal gel form
GEL ELECTROPHORESIS BRIEF PROCEDURE
1. Dissolve the agar, cool the solution, and pour the gel.
Combine agar and water. Bring the mixture to a boil and heat until the agar is dissolved.
Cool the agar until you can comfortably touch the flask.
Place tape across the ends of the gel form (if needed) and place the comb in the form.
Pour cooled agar into the form; the bottom 1/3-1/2 of the comb should be immersed.
When the agar has solidified, carefully remove the comb. Remove the tape (if used) from the ends of the gel form.
When the gel is being prepared a chemical is added to it, this chemical will bind to the DNA and is visible under UV light. The DNA molecules will then appear as series of bands on the gel.
2. Load samples in the wells in the gel.
Make a written record of which sample you will load in each well of the gel.
Place the gel form on a black or dark surface to help you see the wells in the agar.
DNA is added into wells at the top of a pre-made gel. The gel is submerged in a tank of liquid. A power pack can be attached to the tank.
3. Place the gel in the electrophoresis chamber with the wells closest to the negative (black) electrode.
Gel electrophoresis works by using an electric current to pass DNA samples through a gel. The gel acts like a sieve allowing smaller DNA molecules to migrate through the gel quicker than larger molecules.
4. Prepare the salt solution and add it to the chamber.
Add salt to tap water and swirl it to dissolve.
Fill each half of the chamber, adding solution until it is close to the top of the gel.
Then gently flood the gel from the end opposite the wells to minimize sample diffusion.
5. Place the lid on the chamber and connect the electrode leads to the power supply.
Connect the black lead to the negative terminal and the red lead to the positive terminal.
6. Turn on the power supply and adjust the voltage to 50-100 volts.
7. Run the gel for 5-10 minutes.
You will be able to observe the samples separating into different colors.
An electric current is applied across the gel. As DNA is negatively charged it will move from the well towards the bottom of the gel. The gel acts like a sieve, separating different DNA molecules according to their size, as smaller DNA molecules will be able to move through the gel quicker than larger molecules.
8. Turn off the power supply, disconnect the electrode leads, and remove the chamber lid.
9. Remove the gel from the electrophoresis chamber.
Record and evaluate the results of the electrophoresis.
The gel is then visualised under UV light where DNA samples appear as bands. Bands which have migrated further down the gel are smaller than those which have not moved as far.
The image seen under UV light is photographed so it can be analysed. A series of DNA bands will be visible on the gel, and how far they have migrated through the gel will depend on their size.
10. Clean up. Discard the gel in the trash and pour the salt solution down the drain. Rinse the electrophoresis chamber and gel form with tap water; turn them upside down to dry.
The vast applications of electrophoresis are most evident in the health or medical industry, including antibiotic and vaccine analysis. Protein and DNA analysis are also important electrophoresis applications. Aside from allowing researchers to map and see the differences in the genetic code of species on earth, electrophoretic DNA analysis also provides a reliable tool for forensic investigations.
The movement of charged molecules is called mobility. The movement of molecules is towards the opposite charge, for instance, a protein molecule with negative charge moves toward the positive pole of the support medium. The medium may be a paper, gel or a capillary tube.
Vaccine Analysis
Vaccine analysis is one of the many important applications of electrophoresis. There are several vaccines that have been purified, processed and analyzed through electrophoreses, such as the influenza vaccine, hepatitis vaccine, and polio vaccine. The exact steps are done in the vaccine analysis, however, cannot be determined due to confidentiality reasons of the pharmaceutical companies. Nevertheless, data reports from vaccine manufacturers such as Wyeth, Merck and Sanofi-Aventis presents electrophoresis as an effective vaccine analysis method.
"Sterile Round Foam Buccal Swab - Arrowhead Forensics." Sterile Round Foam Buccal Swab - Arrowhead Forensics. Web. 11 Nov. 2015. <http://www.crime-scene.com/store/A-701.shtml>.
"Eppendorf Tube 5.0 ML and Tube Rack 5.0 ML." Red Dot 21. Web. 11 Nov. 2015. <http://www.red-dot-21.com/design/eppendorf-tube-5-0-ml-and-tube-rack-5-0-ml/>.
"Robot Check." Robot Check. Web. 11 Nov. 2015. <http://www.amazon.com/Scilogex-MicroPette-Single-Channel-Adjustable-Pipettor/dp/B0079MASIQ>.
"Eppendorf 022620700 | 5427 R Refrigerated Microcentrifuge with 48 X 1.5/2mL Aerosol-Tight QuickLock Rotor & Lid, Temperature Range: -11 to 40°C, 120V." Eppendorf 022620700. Web. 11 Nov. 2015. <http://www.capitolscientific.com/Eppendorf-022620700-5427-R-Refrigerated-Microcentrifuge-with-48-x-1-5-2mL-Aerosol-Tight-QuickLock>.
"Lysis Buffer #1662002EDU." Lysis Buffer #1662002EDU. Web. 11 Nov. 2015. <http://www.bio-rad.com/en-cn/sku/1662002edu-lysis-buffer>.
"Google." Google. Web. 11 Nov. 2015. <https://www.google.com/search?noj=1&tbm=isch&sa=1&q=warm water bath chemistry&oq=warm water bath&gs_l=img.1.1.0l2j0i24l8.7935.13433.0.15455.15.12.0.3.3.0.153.1418.3j9.12.0....0...1c.1.64.img..0.15.1434.J1bLLmHMGIM#imgrc=ajYdK8Tyj4Y9aM:>.
"MG Chemicals 824 99.9% Isopropyl Alcohol Liquid Cleaner, 125 Ml Bottle, Clear." Amazon.com: : Industrial & Scientific. Web. 11 Nov. 2015. <http://www.amazon.com/MG-Chemicals-Isopropyl-Alcohol-Cleaner/dp/B008UH4AI8>.
"Mouth Tissue - Google Search." Mouth Tissue - Google Search. Web. 11 Nov. 2015. <https://www.google.com/search?q=mouth tissue&rlz=1C1GGGE_enUS594US614&es_sm=122&biw=1444&bih=775&source=lnms&sa=X&ved=0CAUQ_AUoAGoVChMI8veE2YKJyQIVQsFjCh3HuwWO&dpr=1>.
"Google." Google. Web. 11 Nov. 2015. <https://www.google.com/webhp?sourceid=chrome-instant&ion=1&espv=2&ie=UTF-8#q=electrophoresis>.
Web. 11 Nov. 2015. <http://www.colorado.edu/Outreach/BSI/pdfs/electrophoresis_teacher.pdf>
And now we know!
The movement of charged molecules is called mobility. The movement of molecules is towards the opposite charge, for instance, a protein molecule with negative charge moves toward the positive pole of the support medium. The medium may be a paper, gel or a capillary tube.
Vaccine Analysis
Vaccine analysis is one of the many important applications of electrophoresis. There are several vaccines that have been purified, processed and analyzed through electrophoreses, such as the influenza vaccine, hepatitis vaccine, and polio vaccine. The exact steps are done in the vaccine analysis, however, cannot be determined due to confidentiality reasons of the pharmaceutical companies. Nevertheless, data reports from vaccine manufacturers such as Wyeth, Merck and Sanofi-Aventis presents electrophoresis as an effective vaccine analysis method.
Works cited:
Web. 11 Nov. 2015. <http://www.colorado.edu/Outreach/BSI/pdfs/electrophoresis_teacher.pdf>
"Gel Electrophoresis." Gel Electrophoresis. Web. 11 Nov. 2015. <http://www.geneticseducation.nhs.uk/laboratory-process-and-testing-techniques/gel-electrophoresis>.
"Gel Electrophoresis Procedure - Google Search." Gel Electrophoresis Procedure - Google Search. Web. 11 Nov. 2015. <https://www.google.com/search?q=gel electrophoresis procedure&rlz=1C1GGGE_enUS594US614&espv=2&biw=1445&bih=775&source=lnms&tbm=isch&sa=X&ved=0CAcQ_AUoAmoVChMI1Ofd3Z6JyQIVVC-ICh35cAgU#imgrc=8aqQ1svoc67YOM:>.
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