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Transcript
EXTRACTION OF DNA
Grade Level: Late High School
Subject: Molecular Biology
Prepared By: Laura Sedivy and Pat Wagner
Overview and Purpose:
Illinois Learning Standards Addressed:
DNA extraction through centrifugation and precipitation introduces students to current molecular
12. A.4a Explain how genetic combinations
biology techniques and different ways that scientists study DNA. DNA extraction is the first step in most
produce visible effects and variations among
biotechnology procedures, such as gene cloning, DNA mapping, and DNA fingerprinting. This lab activity is
physical features and cellular functions of
also a way to turn an abstract idea/image, such as DNA, into something real, especially for students just
organisms.
beginning to learn about DNA and its structure and function. Extracting DNA helps students understand the
properties of DNA, which in turn lead to its function. In the long run this lab activity should spark the
12. A.4c Describe processes by which
interests of students with respect to what DNA is as well as how and why it is studied.
organisms change over time using evidence
from comparative anatomy and physiology,
Resources:
embryology, the fossil record, genetics and
Brady, Jennifer. Extracting and Analyzing Our Own DNA. The University of Arizona,
biochemistry.
General Biology Program for Secondary Teachers. 2002. Web. 23 Nov. 2010.
Harmon, A. (2008, February 24). The DNA Age: Insurance Fears Lead Many to Shun DNA Tests. [Electronic
version]. The New York Times, 1-4.
Hermanson-Miller, Ingrid and Woodrow, Melissa. Genes in a Bottle Kit DNA Extraction Module. Bio-Rad
Laboratories, Inc. 23 Nov. 2010. PDF file.
Objectives:
At the completion of this lab, TSWBAT….
1. properly use a centrifuge
2. understand how the centrifuge isolates the needed material from the solution
3. derive a connection from their own DNA as to how the structure relates to the function of DNA
4. analyze how this process helps geneticists learn about DNA
Additional Notes:
13. A.4c Describe how scientific knowledge,
explanations and technological designs may
change with new information over time (e.g.,
the understanding of DNA, the design of
computers).
EXTRACTION OF DNA
Background Information:
Deoxyribonucleic acid (DNA) is a nucleic acid that is present in humans and almost all other living
organisms (Hermanson-Miller and Woodrow 8). DNA holds the genetic information that is inherited
generation to generation. This genetic information is stored as a code made up of four bases: adenine,
guanine, cytosine, and thymine. DNA resides in almost every cell of an organism’s body; most DNA is
located within the cell nucleus, but some is also located in the mitochondria (Hermanson-Miller and
Woodrow 8). At the molecular level, DNA resembles a twisted ladder; this structure is called the DNA
double helix (Hermanson-Miller and Woodrow 9). This lab activity makes DNA visible for students. The
DNA extracted by students is precipitated into a large enough mass to see (Hermanson-Miller and
Woodrow 9). Precipitated DNA looks like long, thin white pieces of thread. Since these pieces of thread
are visible when piled together in a large mass, students have the opportunity to get up close and
personal with their own DNA.
Students will begin this lab activity by collecting their own DNA from their cheek cells with a saline
solution (Brady). The cheek cells are separated from the saline solution by centrifugation. A pellet of
cheek cells will form on the bottom of the tubes and the saline solution will be poured off. Then the cells
will be split open with the addition of lysis buffer, which breaks apart cell membranes and maintains the
pH of the solution. The cheek cells will then be incubated in a hot water bath, which destroys enzymes
that break apart DNA. Finally, the DNA will be separated from other cell contents and precipitated with
the addition of cold ethanol (Brady). Students will then be able to study their own precipitated DNA and
become more familiar with its appearance and structure. As students become acclimated with the DNA,
they can begin to connect the structure of DNA to its function.
This lab activity is best suited for a high school Biology II or an AP Biology course. Since this lab is
aimed toward more advanced high school biology, target grades include the 11th and 12th grades. This lab
activity would be best for a class which is doing either a genetics or microbiology unit, since both discuss
DNA.
Set-Up Instructions:
Before this lab activity can be carried out, all of the solutions will need to be acquired or prepared.
A saline solution does not need to be prepared since Gatorade will be used. Also, lysis buffer does not
need to be prepared, since it will be included in the kit. The NaCl salt solution and ethanol solution need to
be prepared in the specified amounts. These solutions should be readily available or can be easily made. If
the salt solution is not available, it can be made using the following recipe:
5 M Sodium Chloride (NaCl):
Additional Notes:
Materials Included in Kit:
1) One Centrifuge
2) Centrifuge Tubes (15 mL)
3) Centrifuge Tube Rack (15 mL)
4) One Hot Water Bath
5) Lysis Buffer (50 mL)
EXTRACTION OF DNA
1) 29.2 g NaCl
2) Distilled water
1. Dissolve 29.2 g of NaCl in 80 mL of water over low heat using a magnetic stir bar.
2. This is almost a saturated solution. It will take a long time for the NaCl to dissolve completely.
3. Bring final volume to 100 mL
Also, all equipment should be set up and ready to use during the lab. Equipment Instructions for
the centrifuge and hot water bath will be included with the equipment.
Before the lab activity, all of the solutions and materials should be laid out and ready for each
group of students to save time. Each group should be provided with small quantities of the solutions in
labeled plastic tubes for use during the lab. About 5 to 10 mL of each solution should be suitable for each
group. 15 mL of Gatorade should be given to each student in sterile plastic cups.
Activity Timeline:
Prior to the beginning of class, the teacher will need to complete what is outlined in the Set-Up
Instructions. The materials list is suited for 12 groups per class with 2 to 3 students per group. Also, since
the centrifuge holds six 15 mL tubes, all of the groups can have their tubes centrifuged in two cycles when
necessary. This lab activity is designed to take place over two 50 minute class periods.
Day 1:
To begin the extraction, one student in each group will need to vigorously swish 15 mL of Gatorade
inside of their mouths for about 30 seconds. Students will then spit the Gatorade (along with the cheek
cells collected) into a small paper cup and then pour it into a 15 mL tube. Students should label the tube
with their initials or group name/number. The teacher will then take six of the students’ tubes and place
them in the centrifuge for three to five minutes until a cell pellet has formed on the bottom of the tube.
The tubes will be taken out of the centrifuge and the Gatorade will be poured out, leaving the cell pellet in
the tube. Subsequent mouthwashes will be completed by the other students in the group and added to the
tube with the cell pellet from the first mouthwash; the steps mentioned above will be repeated.
Once the tubes are done centrifuging and the cell pellets are built up, 2 mL of lysis buffer will be
added to each tube and mixed in by carefully flicking the tube. At the end of the class period, the tubes will
be moved into the hot water bath; they should be incubated at 65-70 degrees Celsius. The tubes will sit in
Additional Notes:
Consumables:
1) Light Colored Gatorade (750 mL)
2) Small Paper Cups (i.e. Dixie cups)
3) Disposable Pipettes (1 mL)
EXTRACTION OF DNA
the hot water bath overnight.
Day 2:
The tubes will be removed from the hot water bath; they should then be flicked to break up the
cell pellets. The tubes should be clear before proceeding. One mL of NaCl will be added to each tube and
mixed well by flicking the tubes for about one minute. The tubes will then each be spun in the centrifuge
for 5 to 10 minutes on high speed. Students will then transfer the clear liquid into a new tube with a
pipette, leaving the precipitate in the centrifuge tube. The students will then add 4 mL of cold ethanol to
the clear liquid in the new tube. The students will then slowly invert the tubes about 5-10 times to
precipitate the DNA out of solution.
Safety Concerns:
Students should wear gloves and protective eyewear. Precaution should be taken when working
with the solutions. If any of the solutions come in contact with students’ eyes, the eyes should be flushed
with water for 15 minutes (Hermanson-Miller and Woodrow 7).
Additional Notes:
Additional Materials Needed:
Items provided by the teacher:
1) Waste Container
2) Permanent Markers/any writing utensils
3) Graduated Cylinders (15 mL)
4) 5M NaCl Salt Solution (40 mL)
5) 95% Ethanol Solution (75 mL)
6) Plastic Tubes for Solutions
7) Napkins (if necessary)