Materials and Methods
Equipment
- 90% isopropyl alcohol
- 100mL graduated cylinder
- dishwashing soap solution
- test tube rack
- Test tubes for centrifuge
- Centrifuge
- 500mL beakers
- Salt (NaCl)
- pipets
- coffee filters
- fruits (papaya, strawberry, banana)
- Plastic zip-lock bags
- Electronic balance beam
- Glass stirring rods
Procedure
- Measure two 30 gram samples of each fruit.
- Place a 30 gram sample of a fruit in a plastic bag and crush the fruit until it has liquefied. This is to break the contents of the cells.
- Add laundry detergent and table salt (NaCl) to the fruit in the plastic bag. Mix by hand. The NaCl makes DNA clump, while the detergent dissolves the nucleus (by dissolving lipids).
- Place a coffee filter over a 500 mL beaker and pour the fruit/detergent solution onto the filter.
- Wait until all liquid has been filtered into the beaker.
- (do only with trials involving centrifuge): centrifuge the solution for two minutes. Pour the supernatant fluid into a new graduated cylinder.
- If centrifuge is not used, pour contents of the beaker into a graduated cylinder.
- Add 40 mL isopropyl alcohol to the graduated cylinder.
- Wait for five minutes until a precipitate (DNA) appears.
- Record the mass of the glass stirring rod.
- Carefully wind the DNA in the solution around the stirring rod, making sure to extract all of it.
- Record the mass of the stirring rod, with the DNA. The difference is the amount of DNA extracted.
- Repeat the procedure five more times, being sure to use one sample of each fruit without the centrifuge and one sample of each with the centrifuge.
Data:
Extraction Without Centrifuge
Extraction With Centrifuge
Calculations
To determine amount of DNA extracted, I subtracted the mass of the stirring rod from the mass of the stirring rod with the DNA. For example: 57.5 – 52.1 = 4.3, so 4.3 grams of DNA were yielded.
The average amount of DNA yielded for each method (with and without centrifuge) was also calculated by adding the amount of DNA extracted from each sample and dividing it by 3. For extraction without centrifuge: (4.3 + 4.7 + 4.9)/3 = 4.433.
Conclusion:
Extraction with the use of a centrifuge yields more DNA than extraction without a centrifuge due to the fact that the centrifuge better separated the contents of the cell, allowing all DNA to collect in the supernatant fluid. This supported my hypothesis that a centrifuge would yield more DNA.
The papaya yielded more DNA than the banana and the strawberry. This supports my hypothesis that the papaya would yield the most DNA. This is due to polyploidy—as the most genetically advanced fruit, the papaya has the most number of chromosomes.
Evaluation
Source of error:
The coffee filter may not have allowed all liquid from the fruit to pass into the beaker. Its purpose was to keep the solids separated from the liquids, but some liquid remained on top of the filter with the solid. This amount may vary from trial to trial, giving us inconsistent results. Also, DNA could not be extracted on the rod without getting some fluid on the rod, which would add to the mass of the rod with the DNA. The DNA itself contained fluid, which would have added to the mass. Not all of the fruit could be liquefied by mashing it by hand, so not all cells were broken and thus not all DNA was extracted.
Statement of improvement:
This lab could be improved by using a better filtering system. The coffee filters left too much liquid with the solid it strained. Perhaps a stronger filter would be better, as the remaining solid could be pressed and yield more liquid. The DNA invariably contained fluid when it was extracted, so drying the pure DNA before recording its mass would remove that extraneous mass. Utilizing a blender or another more efficient method of pulverizing the fruit would yield more DNA, as it would break all cells.
Learning Statement:
What I learned from this experiment was that the inclusion of one additional step (with the centrifuge) could have a profound effect on the amount of DNA yielded. I learned that the centrifuge separates the contents of a solution far more effectively than letting the solution sit. I also learned that polyploidy plays a role in the amount of DNA contained in each fruit, as the fruit with the most chromosomes yield the most DNA. This experiment shows the importance designing an experiment with the right procedures, as the omission of one step produced a much lower percent yield of DNA.
