DNA Extraction 101

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by ClarABC
Last updated 5 years ago

Discipline:
Science
Subject:
Experiments
Grade:
9,10,11,12

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DNA Extraction 101

DNA Extraction 101

Why are we making onionsauce?The onions were placed in a petri dish and strenuously sliced until the onion resembled an applesauce, or smoothie composition. (This process was aided by the addition of water, at incremental points during the cutting process). By cutting the onion we are effectively breaking apart the cell walls of the onion cell, hence exposing the deoxyribonucleic acid (DNA). Or the cell walls are exposed, which may then be later broken down by the extraction /buffering solution (specifically, the soap) (Cavalcade Publishing, 1999).SO what's the point of an extraction solution then?DNA is found only within the nucleus of a cell for a reason – it simply cannot survive outside the nucleus as it’s proteins will dissolve as a result of the acidity and pH levels of any organism (Cavalcade Publishing, 1999). In this lab we utilize this knowledge, and purposely break down the cell walls of the onion and allow the DNA to dissolve so that we may extract it later on. For this reason salt is used in the extraction solution (increases salt concentration) as well as baking soda (which alters pH levels) (Cavalcade Publishing, 1999). Salt also serves a secondary purpose as it neutralizes the charges on the sugar-phosphate backbone of DNA. The Na+ molecules attach to the negative PO3- groups, effectively neutralizing the DNA as a whole. As a result the PO3- no longer bond with H+ (in H2O), making it less soluble in water, and lose its solubility tendencies once the alcohol is added. (Oswald, 2007). Detergent is also a component of this solution. The molecules that make up the soap have both a hydrophilic and hydrophobic ends, as a results the soap molecules latch on to either/both the proteins and lipids (which make up the phospholipid bilayer). By removing the proteins and lipids, the soap eventually breaks apart the cellular membrane, releasing the DNA (University of Utah, n.d.).

Why do I need to use ethanol if I've already exposed the DNA?Water originally has a high dielectric constant, meaning that it’s hard for the Na+ to combine with the PO3- (Oswald, 2007). However alcohol, has a low dielectric constant, allowing for the salt to bond with the phosphate group (Oswald, 2007).This therefore decreases the soluble nature of DNA, and makes it insoluble in water (Oswald, 2007). This is the reason the DNA floats above the solution that had previously passed through the filter. This shows us that DNA is originally soluble, with a negative charge on its phosphate group, which under normal circumstances would bond with one of the two H+ in H2O. Using cold ethanol is also important, because the cold temperatures protects the DNA strands by slowing down enzymes that may break it apart. The cold also helps to allow the DNA to precipitate when alcohol is added quicker. (University of Utah (b), n.d.)So then what effect does water have on DNA?Prior to the addition of the alcohol, the DNA dissolves in water when stirred. This occurs because the phosphate sugar backbones bond with the hydrogen ions – making DNA soluble in water. However, with the addition of alcohol allowing for thesodium to disrupt and bond with the phosphate group, thereby making DNA an insoluble substance when in water.

WAIT! Can you go over that again?To reiterate, the purpose of:Shampoo: •To break down the cellular membrane by pulling apart the lipids and proteins found in the phospholipid bilayer (University of Utah, n.d.).Baking Soda:•To alter the pH levels, to match those within the body, and enable the DNA to dissolve in water (Cavalcade Publishing, 1999).Salt:•To increase salt concentrations to match those found within the body, and therefore help the DNA dissolve in water (Cavalcade Publishing, 1999).•When ethanol is added, the salt disrupts the bonds formed by the phosphate bonds and hydrogen ions (Oswald, 2007).- Na+ then bonds with the phosphate group to neutralize DNA (Oswald, 2007).- DNA then become insoluble in water (Oswald, 2007).Ethanol:•Lower the dielectric constant, and allow for the Na+ to bond with the phosphate group – therefore making the DNA non-polar or neutral (Oswald, 2007).

So what is DNA like? •DNA has a negative phosphate group and will either bong with hydrogen ions in water to become soluble in water, or with sodium ions in alcohol and become insoluble in water•Extremely long in length, but very thin in diameter•Milky\cloudy in colour•AcidicHow did you know it was DNA?DNA is extremely long – that’s the biggest thing I gathered from this lab. However, while it may be very long it is still almost microscopic in diameter. In fact, my lab partners and I had a real difficulty seeing the DNA once we had spooled and taken it out of the test tube completely. Only in certain lighting could you see the thin fabricated lines of the DNA coiled around the stirring rod. In solution, the DNA was much easier to see, by gently agitating the test tube the layer of DNA floating in between the layer of water and ethanol could be seen (ie. the cloudy layer). The volume /quantity of DNA extracted from such a small portion of the onion also came as a large surprise. DNA is an acid (hence the name deoxyribonucleic acid) and would turn the blue litmus paper red/pink in a perfect world. Unfortunately, this lab is very flawed, as a result the blue litmus paper did not change colour. This may be due to the remnants of the basic extraction solution still on the stirring rod. However, the important thing here is that we understood what should have happened, ie. the litmus paper turning pink/red signifying the presence of an acid, which would then signify the presence of DNA.

And it's filtered because...?Utilizing filter paper while being able to gather and collect a significant amount of DNA, shows us that DNA is extraordinarily small and compact relative the information it stores. It also shows us that in comparison to the other materials found in a cell (ex. ribonucleic acid), DNA is smaller.

Figure 1. Above is an image of the sliced onion. Taken by Chong, C. 2014.

Figure 2. Below is an image of the clustered DNA located between the layer of extraction solution and ethanol. Taken by Chong, C. 2014.

Figure 3. Above is an image of the blue litmus paper test, which yielded negative for acid. Taken by Chong, C. 2014

Figure 4. Above is an image of the extraction solution and sliced onion going through the filter paper. Taken by Chong, C. 2014

In case you forgot how the lab works, here's a short demonstration on extracting the DNA from a strawberry, (we did onion, but the two are very similar) (Sick Science!, 2012).


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