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Purpose:
We intend to use strawberries or bananas and isolate the DNA of either one, we want to understand the layers there are to get to DNA by quite literally going through the layers to get to DNA enabling us for a better understanding of how DNA works and where it is located.
Materials:
Ethanol, 95% denatured, CH₃CH₂OH, 12 mL
Ethylenediaminetetracetic acid solution (EDTA), 0.1 M, 1 mL
Sodium chloride solution, NaCl, 8.0%, 1 mL
Sodium dodecyl sulfate solution (SDS), CH₃(CH₂)₁₁OSO₃Na, 10%, 1 mL
Deionized water or distilled water, H₂O, 40 mL
Banana or Strawberry or both!
Beakers, 50-mL, 2
Filter paper
Glass stirring rod
Graduated cylinders, 25-ml, 2
Ice-bath
Pipets, graduated, disposable, 3
Resealable bag
Test tube, 5-mL
Preparation:
Prepare an 8.0% sodium chloride solution: Dissolve 8.0 g of sodium chloride in about 70 mL of deionized water. Dilute to a final volume of 100 mL with deionized water. Place the solution in a labeled bottle.
Prepare a 10.0% sodium dodecyl sulfate solution (SDS): Dissolve 10.0 g of sodium dodecyl sulfate in about 70 mL of deionized water. Dilute to a final volume of 100 mL with deionized water. Place the solution in a labeled bottle.
Prepare a 0.1 molar ethylenediaminetetraacetic acid solution (EDTA): Dissolve 3.72 g of EDTA in about 70 mL of deionized or distilled water. Dilute to a final volume of 100 mL with deionized water. Place the solution in a labeled bottle.
The 95% ethanol should be ice cold (about 0 °C) when used. Place it in an ice bath before use.
Procedure:
Add 1 mL of the 8.0% sodium chloride solution to a 50-mL beaker. Set the beaker aside.
Pour 40 mL of deionized water into a clean, resealable bag.
Place a one-inch section of banana, or a small strawberry into a resealable bag.
Smash the fruit in the resealable bag until the fruit mixture has a thick liquid consistency. This is what your teeth are responsible for in digestion.
Pour the fruit mixture through four layers of cheesecloth, or filter paper, which is layered on top of a clean 50-mL beaker. Note: Do not squeeze the cloth or filter paper.
Use a clean, graduated cylinder to transfer 10 mL of the filtered fruit solution into the small beaker that contains the 8.0% sodium chloride solution.
Use a clean, graduated pipet to add 1 mL of the 10.0% SDS solution to the beaker. This is to break down the phospholipid cell membranes.
Use a clean, graduated pipet to add 1 mL of the 0.1 molar EDTA to the beaker. This is to inhibit DNase, a molecule that breaks down DNA.
Gently swirl the resulting solution in the beaker
Holding the beaker at a slight angle, use a clean graduated cylinder to carefully transfer 10 mL of ice-cold 95% ethyl alcohol down the side of the beaker so that the ethanol forms a layer on top of the aqueous solution in the beaker.
Carefully place the beaker back on the tabletop, making sure the two layers do not mix.
Allow the beaker to sit for one minute and observe the DNA precipitating out of the solution at the interface between the cold ethanol and the aqueous layers.
Add 1 mL of around 0 °C 95.0% ethanol to a small test tube.
Gently place a clean glass stirring rod into the interface containing the DNA. Collect the DNA by turning the stirring rod in one direction -- the DNA will "wind" itself onto the glass stirring rod, almost like spaghetti.
Carefully remove the stirring rod and DNA from the beaker and transfer the DNA to the small test tube containing the ethanol. Observe the DNA strands floating in the alcohol.
Observations:
1 ML is barley a drop
40ML is not very much
Smashing the fruit is easier than I first thought
We used coffee filters instead of cheesecloth I wonder if that has an effect on the result
It was cool to watch the ethanol form a layer on top of the solution
I could see a cloudy like substance appear in-between the two solutions
DNA is a lot easier to swirl on a stirring rod than spaghetti is on a fork
The DNA is hard to get off the string rod
Data Collection:
I got a "small" amount of DNA it was very little to the scale of me or even ML's but the fact is was visible to my naked eye shows how much I really had. The DNA I had was very clumped up which I thought was normal until I saw some of my classmates, after comparison to some people I saw that theirs was more spread out where ours was more condensed. I wonder what caused it, could it be that we used strawberry's and they have more DNA so its more dense, Or maybe it was human error when trying to get DNA off of the string stick, or it could even be that we stirred it too much!
Conclusion:
In this experiment I learned that there is a lot that goes into keeping the DNA in the nucleus including the cell wall, cell membrane DNase. We have completed the experiment and found that when all the inner workings are kept separate via various chemicals and you are left with DNA both figuratively and literally in the middle. The whole process was really cool to complete and watch.
Personal reflection/discussion:
Next time I do a lab to this caliber I need to be more carful with directions because me and my lab partner had accidently filtered the fruit juice straight into the beaker with the NaCl solution whereas the directions said to put it in an empty beaker then use the graduated cylinder to measure out 10ML and insert it into the NaCl solution. I don't see how this could contribute to the clumping on the DNA but this was the only time we went astray from the directions so it might have contributed to the clumping DNA.
Lab Partner: Cory Torode
Lab Sheet:
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