Professional Documents
Culture Documents
Photo Credit: Milkweed. Digital image. Blue Ridge Discovery Center. N.p., 8 Oct. 2010. Web. 17 Dec.
2014. <http://blueridgediscoveryproject.blogspot.com/2010/08/marvelous-milkweeds.html>.
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Table of Contents
Project Proposal 3
Introduction
Target of Interest
Experimental Overview 4
DNA Sample Collection
DNA Extraction and PCR Amplification
Gel Electrophoresis
Sequencing and Interpretation
Implications and Future Research 5
Possible Timeline 6
Budget 7
Proposed Materials With Sources
Appendix I 8
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Project Proposal
Introduction
Target of Interest
3
Experimental Overview
4
Implications and Future Research
5
Possible Timeline
Week Task
1 Obtain milkweed seeds, order necessary materials, organize three
groups for planting
2 Plant the first set of seeds
3 Perform DNA extraction on first set of seeds.
4 Plant the second set of seeds
5 Perform DNA extraction on second set of seeds
6 Begin Gel Electrophoresis on DNA from seed sets one and two
7 Plant third set of seeds
8 Perform DNA extraction on third set of seeds
9 Finish Gel Electrophoresis on DNA from third set of seeds
10 Compile information and prepare final report
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Budget
Only Materials not accessible through school
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Appendix I
Example Protocol for DNA Extraction adapted
from BIT 210 Molecular Genetics: Lab 1- DNA
Extraction
Protocol for isolating DNA:
1. Record samples of Milkweed 1-15 in a table. (1-5: group 1; 5-10:
group 2; 11-15 group 3)
2. Label 1.5ml flip-top centrifuge tube with the assigned number.
3. Pipet 200µl of lysis buffer into the tube.
4. Weigh 50-100 mg of the plant material and record weight.
5. Use a razor blade or scalpel to cut the material into small pieces
(less that 1-2mm in diameter)
6. For each group, use a clean micropestle to grind the plant material
for at least 3 minutes. Be careful not to spill or splash any lysis
buffer out of the tube. Avoid having plant material compact into the
bottom of the tube. If the material is compacted and/or stuck, use a
clean pipet tip to loosen the mass. Grind the material until the
particles are very fine (difficult to see by eye). This may take
additional time.
7. Once a homogeneous lysate has been generated, add an additional
500µl of lysis buffer. Continue grinding until solution is
homogeneous.
8. Close the microcentrifuge tube securely, and plant in a centrifuge.
Centrifuge at full speed for 5 minutes at room temperature.
9. For each group, label a new microcentrifuge tube with the assigned
number. Add 500µl of 70% ethanol into each tube.
10. Retrieve samples from microcentrifuge and add 400µl of the
supernatant to the 70% ethanol in the appropriately labeled tube,
taking care not to disturb the pellet. Pipet up and down to mix lysate
and ethanol. Change pipet tips for each sample.
11. Label the top edge of a purple mini DNA extraction column for each
plant and place columns in 2 ml capless collection tubes.
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12. For each sample, pipet 800µl of cleared plant lysate into the
appropriate column. Change pipet tips for each sample.
13. Centrifuge columns for 1 min. Discard the flow through from the
collection tube and replace the column in the appropriate collection
tube.
14. Add 700 µl of wash buffer to each column. Spin at full speed for 1
min. Discard the flow through. Repeat two more times for a total of 3
washes.
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