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Project Proposal

⌘ Analysis of Global Warming on the Germination of


Prepared for: Burlington County College Undergraduate Research

Prepared by: Lauren Seitz and Amanda Sharpe, Class of 2015 - Biology
Faculty Mentor: Professor Anne Tokazewski, Biology Department

December 17, 2014

Photo Credit: Milkweed. Digital image. Blue Ridge Discovery Center. N.p., 8 Oct. 2010. Web. 17 Dec.
2014. <>.

Table of Contents
Project Proposal 3
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

Project Proposal

Over the past 20 years, Monarch Butterfly population has decreased by

approximately 90%. Similarly, the Milkweed population, which they live on
and eat, has also decreased drastically. With global warming affecting the
seasons, it is also possible that the effect on the seasons will result in less
milkweed plants and a lower success rate for germination. The research
proposed studies the effects that weather and temperature exposure has
on the germination of Common Milkweed.

Target of Interest

The point of the research is to target the ideal temperature exposure

needed for peak germination of milkweed seeds. This goal is accomplished
by focusing on different environmental stressors and how seeds respond.
Success is determined by the seed’s ability to grow and the success of DNA
gel electrophoresis done to the grown plants.

Experimental Overview

During this experiment, Common Milkweed seeds will be placed in the

freezer for different amounts of time. There will be 3 groups: the first group
will be exposed to cold temperatures for only 2 weeks and then be planted,
the second group will be exposed to cold and warm temperatures over the
course of 4 weeks and then be planted, and the last group will be fully
exposed to cold temperatures for 6 weeks and then be planted. After the
seeds grow, the plants’ DNA will be extracted and Gel Electrophoresis will
be conducted on the leaves to evaluate any genetic differences that may be
observed between the Common Milkweed groups depending on their
exposure to cold temperatures.

Implications and Future Research

If Common Milkweed is shown to be affected by the exposure to cold

temperatures, further research can be conducted on different species of
milkweed. The different species may have different preferences that would
make them more fit for the environmental conditions that exist in New
Jersey. Knowing these preferences would allow for conservationists to plant
the ideal milkweed with the intention of increasing the Monarch Butterfly

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

Only Materials not accessible through school

Proposed Materials With Sources

Description Quantity Unit Price Cost

100 Common Milkweed Seeds 2 $2.00 $4.00

Total Costs = $4.00

Appendix I
Example Protocol for DNA Extraction adapted
from BIT 210 Molecular Genetics: Lab 1- DNA
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
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.

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
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