DNA Lab

Onion and E. coli DNA Extraction

Materials | Procedure, Observations, and Analysis | Ending Observations  Error Analysis | Back to DNA Extraction Main Page

Materials:

  • Onion, medium sized (or 5g Escherichia coli in a liquid growth medium for other lab)
  • Knife
  • Balance scale
  • Blender
  • Ice Bath
  • Glass Rod
  • 2 250 mL beakers
  • 2 250 mL Erlenmeyer flasks
  • 1000 mL beaker
  • 500 or 600 mL beaker
  • 10 mL graduated cylinder
  • 100 mL graduated cylinder
  • Cheesecloth
  • Thermometer
  • 80 mL homogenizing medium
  • 80 mL ice cold 95% ethanol
  • 10 mL chloroform

 

Procedure: 

The procedure, observations, and analysis are next to each other below.  On the left is the procedure, followed by the observations and analysis.  All are for the onion extraction.  Any differences are mentioned, but with no mentioned differences, the assumption is that there are none.

Procedure

Observations

Analysis

Dice 50 g of onions into cubes smaller than 3 mm, and put them in a 250 mL beaker

The onions were already cut

Increasing the surface area of the onions, which cutting them in smaller pieces does, helps to make the membrane at the surface easier to dissolve, and also allows for more efficient absorption of heat and solutions

Add 80 mL of homogenizing medium to the diced onions.

The onions were spread evenly and densely pached, but floating in the medium.  The bacterial solution smelled like it was viable when wafted.

The homogenizing medium, made of SLS, sodium laurel sulfate, and EDTA, was similar to a detergent.  It helped break up the phospholipid bilayer of the cells' plasma membranes and nuclear envelopes.  When in SLS, the proteins and lipids of the membrane, but especially the lipids, are broken down because SLS causes the bonds holding the membrane together to break.  The lipids, which are repelled by the SLS, separate and break up the membrane.  This is why detergent is used to remove a grease stain; grease is a lipid.  The EDTA helped weaken the membrane as well, making the DNA that was in the nucleus now available for extracting. 

Incubate the beaker in a  

60 C water bath for 15 minutes

We had trouble controlling the temperature.  It was at 55 C for most of the time.  The 5 mL of ecoli that we used turned the homogenate into a white/very light yellow color.  The temperature was easier to control.  The onions became noticeably softer

The incubation was used to speed the breakdown process by energizing the molecules, and also, hopefully help to dissolve the phospholipid bilayer by destroying the proteins and breaking the bonds that hold the phospholipids in place.  By exciting the molecules, the heat might cause the fluid bilayer to break and become a gas-like substance, no longer serving as a barrier for the DNA.  The heat softens the membrane as a whole.  This entire step, homogenation, is used to free the DNA from its protective barriers

Cool the preparation to 15-20 C in an ice path

The temperature of the beaker cooled from 55 to 25 in about seven minutes

Cooling the solution will help prevent denaturation, which might destroy the DNA if it was exposed to prolonged heating because of abundant DNAases

Pour the cool preparation into a blender and fasten lid.  Homogenize for 45 seconds at low speed and 30 seconds at high speed [OMIT FOR BACTERIA]

It turned into a thick, opaque, foamy white solution, like a piña colada or the foamy part of detergent.  We skipped this step with the E. coli-once it reached 22 degrees, we left it in for 15 more minutes

This also frees the DNA from another protective barrier, the thick cell wall that all plant cells have.  The blending should cause many of the cell walls, containing cellulose, to break, and therefore leave the DNA in the cytoplasm, and no protective thick cellulose wall blocking the cytoplasm from its external environment.  We did not need to do this to the bacteria because it does not have a cell wall

Pour the homogenate from the blender into a 1000 mL beaker.  Allow it to sit in ice for 15-20 minutes

Only 2/3 of thew foamy solution (which reached 700 mL) was in ice.  After 17 ½ minutes the solution really condensed.  There was a much smaller layer of foam at the top and a light yellow liquid at the bottom.  The E. coli was still a whitish yellow.  There was a milky white layer at the top

This is simply for storage, but also to cool the solution for its later contact with ethanol

Filter the homogenate through the cheese cloth, being sure to leave the foam behind, in the 1000 mL beaker

A very light yellow liquid, like lemonade, filtered into the 250 mL beaker.  We dropped the cheese cloth in the beaker and were forced to start over.  Only a few bubbles were in the beaker.  We squeezed very hard and some foam entered, but most stayed in the cheese cloth, which we returned to the beaker.  We then put it on an ice bath.  After we filtered, the E. coli was a milky white, but when swirled, there were clear patches

This leaves behind thicker materials, including any parts of the remaining onion itself.  The liquid that is filtered through contains the DNA, no longer held behind a plasma or nuclear membrane, and ready to extract.  All waste not needed for the experiment is filtered out, such as cellulose of the cell walls, pectins, and excess tissues like the skin sclerenchyma cells of the onion

Put the alcohol in the freezer

 

The ethanol, as later explained when it is used, must be ice cold to separate the DNA

Place in ice bath for 15 minutes

The onion began to separate into layers. Most was a white foam with sclerenchyma tissue suspended in it.  At the bottom of the beaker was a pale yellow liquid.  The baterial cells condensed into large white masses of cells and homogenizing medium

Cooling

Pour 50 mL of the filtered homogenate into a 250 mL flask.  Put on aprons and goggles.

Almost all of the bubbles had disappeared, but there were granules of some substance at the top

We need only part of our solution in this part of the experiment, and we simply moved it to a smaller container.  The aprons and goggles were a safety measure because we were working with chloroform, a carcinogen

Using the 10 mL graduated cylinder, add 2 mL of chloroform to the homogenate, pouring it down the side of the beaker

We carefully poured about the right amount of chloroform down the side.  The solution was on the top, then a protein interface, and then the chloroform, a much lighter color, at the bottom.  It was hard to see the interface, which was a whitish color

Chloroform is organic and hydrophobic, and because there is a rule of science that like dissolves like, the chloroform will dissolve other organic and hydrophobic, or nonpolar, molecules.  It helps dissolve the inner proteins of the DNA because inner proteins of a polypeptide with tertiary structure are the ones with the most hydrophobic "R" groups.  The denaturation of these histones, as well as other structural proteins and proteins in the plasma membrane, helps the DNA uncoil and makes it easier to extract.  The proteins, with only their hydrophilic shells remaining, precipitate out of the solution and form their own layer at the bottom, between the homogenate that it has precipitated out of, and the chloroform, which it is no longer soluble in. Only the bottom proteins would precipitate out, but the chloroform must get to the bottom by passing through the homogenate, and dissolves and moves proteins to the bottom as it quickly forms it own layer.  In fact, in the medical industry, chloroform was used to dissolve organic materials before it was listed as a carcinogen and banned in 1976.  It is very dense, with its vapor being four times denser than air, which is why it's layer was under the homogenate layer.

Gently swirl the contents of the flask

We didn't do this every time

This gives a greater area of contact between the chloroform and homogenate, helping dissolve and remove proteins.

Pour the homogenate into another, clean 250 mL flask, leaving the chloroform and protein layers.  Rinse the flask with the chloroform and protein layers.

It was hard to pour exactly.  We erred on the side of not pouring all of the homogenate rather than pouring some of the chloroform in,  Therefore, no chloroform should have stayed in

The proteins are not needed for the rest of the experiment, and would in fact get in the way of extracting DNA by helping hold the DNA in tight coils, so it is advantageous to remove the proteins from the solution.

Repeat the last three steps, beginning at adding the chloroform, four more times.  By the fifth time, there should be much less of a protein interface

There was definitely less interface every time we did this, in both E. coli and the onion

The more times this is done, the more proteins that will be removed.  Therefore, it was better to get out as many proteins as possible

Gently pour the homogenate into a 250 mL beaker.  Make sure not to contaminate it with chloroform.  You may need to leave a small amount of homogenate behind

We did leave a little behind

Just the ending of our fifth chloroform transfer

Place the beaker with its deproteinized homogenate in an ice bath and let it cool until it reaches 10-15 C

It cooled only one or two minutes, but may not have even been quite at 15 C in both cases

Both the ethanol and the homogenizing mixture must be cooled because DNA is only not soluble in ethanol when both are at very cool temperatures

Slowly add ice-cold ethanol down the side of the beaker until the white, stringy DNA precipitate appears.  It may not take all 80 mL

Before we poured, there was a film at the top of the solution.  We poured in all 80 mL of ethanol.  There were 3 clear layers; a clear ethanol layer at the top, a DNA interface, clear with whitish strands, and quite visible unlike the protein, and a yellow homogenate at the bottom *for observations on the E. coli, see below

The DNA is polar, but the reaction with the ethanol makes the DNA nonpolar, and therefore resistant to the homogenizing medium that it is in.  The DNA forms its own layer, a precipitate between the liquid ethanol and liquid homogenate.  Also, the DNA is the only component of the solution that is not soluble in the ethanol.  Therefore, it becomes its own clear layer on top of the lighter ethanol, but free of the soluble homogenizing medium. However, the reaction above, with the DNA precipitating to the top of the homogenate because it becomes nonpolar and remaining under the ethanol because it is not soluble only happens at the top of the solution where the DNA is exposed to the ethanol and does not move far to precipitate out.  Therefore, the top DNA  remains between the two layers.

Spool out, or wind up, the stringy DNA onto a glass rod by rotating the rod in only one direction.  Continue to rotate the rod as it moves in large circles

The DNA was clear and easy to get out.  We got a lot in the Epindorf tube, but it was slightly hard to get the DNA off of the rod

The glass rod was helpful because the negative charge of the phosphate groups in DNA was attracted to the positive silica in the glass.  It was necessary to spool the DNA rather than try to pull individual pieces up because the DNA was very fragile and broke when this was attempted

To store the DNA, ensure that it is covered with ethanol.  Then, gently push the DNA off the glass rod into the an Epindorf tube

The DNA is covered with ethanol and stored in the freezer.  The extraction took 1 hour, 45 minutes

The onion extraction was a success.

*E. Coli and ethanol

After pouring all of the ethanol in, there were two distinct layers, the ethanol and the homogenate, but no interface with DNA.  We tried to centrifuge it, but we still saw only a clear solution, no white wisps.  This extraction failed, our only one that did not work.

For a full explanation of why the E. coli extraction was not successful, see the error analysis.  In short, this experiment was designed for onions, and basic flaws prevented the extraction of E. coli DNA.  To learn about the centrifuge process, see the next lab.

Ending Observations: 

We had the easiest time removing the DNA from the onions, but we were unsuccessful in the DNA extraction from the E. coli.  All other observations are above, in the middle column.

Conclusions:  This experiment taught us the process of extracting DNA from both plant and bacteria cells.  DNA extraction is a key process in science.  To extract DNA, three things need to be done.  The DNA must be accessible (homogenization), the DNA must uncoil and become extractable, as well as separate itself from the proteins (deproteinization), and the DNA must become a visible, individual layer and therefore be able to be extracted (precipitation). 

In homogenization, to make the DNA accessible, all cell coverings must be removed so that the DNA floats freely in the solution.  There were three of these; the cell wall, the cell membrane, and the nuclear envelope.  The cell membrane and nuclear envelope are both made of phospholipid bilayers, with embedded proteins.  We used sodium laurel sulfate and EDTA to break the bonds of the phospholipid bilayers, therefore allowing the transport of DNA.  The tougher cell wall, made of cellulose, was torn apart by the powerful blender.  The proteins in the cell membranes were taken apart by the chloroform, but this chemical was more important to deproteinization. 

Once the DNA was free in the solution, it needed to be unraveled so it grew and became more accessible.  We were able to do this by using chloroform.  The chloroform took apart the histones that accounted for the tight structure of DNA, and therefore it was only the DNA that made up the chromosomes, which were formerly comprised of half proteins.  The chloroform ate away at the proteins from the inside because it dissolved the most hydrophobic amino acids first.  The proteins dissovled, leaving DNA in an extractable form.

The final step in the process was precipitation.  This was done to make the DNA actually visible and extractable.  The ethanol created a new level of DNA by making the DNA at the top of the homogenate nonpolar, and which made it repel the homogenate.  The DNA was not soluble in the ethanol, so it formed its own level, and our experiment was complete.

In this experiment, we learned many things.  We learned practical scientific uses of SLS, EDTA, chloroform and ethanol.  We were able to make very precise measurements and learned to analyze different steps of an experiment in order to figure out why each step was performed.  But most importantly, we learned the process of DNA extraction.  This process is becoming more and more important, especially with gene insertions that are being used to treat diseases now.  This has been a headline every day this week.  To insert DNA, it is necessary to extract it first.  Therefore, we learned a process that will stay with us and that we should repeat many times throughout our scientific careers.

Error Analysis: 

In our onion extraction, we seemed to be successful, but we could have probably extracted more DNA if we had been more precise.  For instance, we had trouble controlling the temperature of the water bath, and we did not keep our solution in the water or ice baths for exactly the right amount of time.  This could have meant the denaturation of some of our DNA.  We were able to see a lot, but did not get very much, a fault in our spooling skills, but also a fault in our conduction of the experiment to that point.

However, our huge error occurred when we were not able to extract the DNA from the E. coli.  The temperature in the water bath was too high for some time, but it is doubtful that are problem was in the denaturation of the DNA.  Even if some of the DNA had denaturized, we still should have been able to extract some, but there was none apparent, even after centrifuging.  One thing that might have accounted for this problem was that we saw a mass of cells in the beaker after the chloroform part of the experiment.  Therefore, the cells had not come apart.  When this happened, the ethanol could not have reached the DNA and "coaxed" it to the top.  We have one possible reason why this might have occurred.

The biggest problem with the E. coli extraction was that this lab was not designed to extract DNA from E. coli.  Therefore, there were probably steps that would be needed to break up the E. coli cell for the DNA to be available for extracting.  Because E. coli is a prokaryote, there is no nucleus, and no nuclear envelope that is needed to be broken down.  We believe that the SLS and EDTA still worked on the cell membrane, but the protective covering that is not accounted for is the cell capsule, a covering of all bacteria.  Blending broke the cell wall of the onion, but we did not put the E. coli in a blender.  Therefore, the cell capsule and the cell wall were never broken, and therefore the entire rest of the experiment was unsuccessful.  This is why there were intact cell masses that we saw after the chloroform test.  If we were to repeat this experiment, we would at least need to find a way to break the cell capsule and cell wall, but we should find a procedure that is actually specified to extract DNA from E. coli.

 

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