Craig_Bridge
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Postby Craig_Bridge » Wed Oct 17, 2007 4:21 pm

I've done some searching and found http://www.usc.edu/CSSF/History/2004/Projects/J0404.pdf and http://www.geocities.com/CapeCanaveral/Hall/1410/lab-B-11.html which appear to be similar extraction proceedures using Isopropyl alchohol. This means that the chemical difference and any solubility differences between alchohols isn't important for this separation step.

In looking back through this thread, you, the researcher, have not stated what your hypothesis is. Several responses by experts have hinted at some potential areas that might be interesting. Without a stated hypothesis, we are not going to be much help in identifying controls and variable types. What you have so far is a demonstration "Can I extract Onion DNA using ... process?". Proving that what you extracted is DNA is a much more difficult problem, but even that does not appear to be the kind of a science project that your teacher wants you to do because it is just a demonstration.

You need to go back and try to understanding the scientific method http://www.sciencebuddies.org/mentoring/project_scientific_method.shtml and see how you might form a hypothesis that might use this or a similar extraction process to answer a scientific question that will satisfy your teacher's criteria. Doing demonstrations is usually much easier than applying a scientific method to answering a unique question. Repeating somebody elses demonstration is definitely easier yet. It is not that you aren't learning things, but you aren't "doing your own science" in a way that will stand on its own.
-Craig

bryanandsebastian
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Do i need to.............?

Postby bryanandsebastian » Thu Oct 18, 2007 1:51 pm

When I put the chopped onion mixture into the blender do I need to add some water or do I leave it how it is. So then after blending I just put it into the measuring cup with the salt, detergent, and 100ml of dH2O. I want to do this experiment with the blender and without the blender. We made a variation on this experiment and we are comparing the extraction DNA weight of animal cells vs. plant cells. By the way we went to a liquor store and there was no everclear ( we live in FL ) so we're going with the isopropyl. The teacher approved.


Thanks,
Bryan
Bryan
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Sebastian
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Craig_Bridge
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Postby Craig_Bridge » Thu Oct 18, 2007 6:04 pm

When I put the chopped onion mixture into the blender do I need to add some water or do I leave it how it is. So then after blending I just put it into the measuring cup with the salt, detergent, and 100ml of dH2O.
You need to start thinking about what each step in the extraction process is actually accomplishing.

"Chopping in a blender" is a mechanical operation that increases the total onion surface area which means there are more onion cells with surface exposure. Note: Some onion cells will burst from the chopping process. The next step is part of breaking down cell walls to let the DNA and other things out of the cell into your solute. The more cells that are on the surface, the more that break apart, and the more of the original DNA you can collect.

If you add water in the chopping step, you will alter the concentration of the salt/soap solution. You might be able to adjust the 100 ml of water to compensate; however, if you are trying to determine a ratio of milligrams of onion and other cells to milligrams of extracted DNA, then adding water in the chopping operation will affect your mass measurements. You are probably going to have to measure the mass of what you get out of the blender because there is probably no way to get all of the onion out of the blender to make use of it. Some onion will be left for the dish washing step.

You definitely need to figure out what substances (like starch) that are in the different cell types you intend to use so that you can modify the extraction steps if needed so they don't contaminate your extracted DNA or interfere with the extraction.

You probably need to use the same extraction steps on all cell types to eliminate any question about using different methods on different cell types affecting your ability to extract all of the DNA.

You probably also have to reprocess your precipitates to determine if you managed to break down all of the cells or if DNA was lost in a step. Use of a motar and pestle to grid the cells in the first precipitate and reprocessing won't be quantitative; however, it should provide some qualitative information and answer the nagging question did you break a high enough percentage of the cells to not have a high experimental error variation.

If you break down different percentages of different cell types, then your idea of measuring ratios of extracted DNA to starting cell mass may render any conclusions meaningless.

You really need to post a hypothesis for us to really help you with figuring out if you have thought through everything before you waste time and efforts.

Florida is one of the states where you can't buy 95% ethanol for human consumption and I am NOT the best expert around here to be helping you. I've just picked up on some of your potential troubles.
-Craig

bryanandsebastian
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THANK YOU!

Postby bryanandsebastian » Fri Oct 19, 2007 1:28 pm

I think that your telling me not to use a blender but chop with a knife, right? :? I got the part about the water and I realized that the volume of water is REALLY important :idea: My dad will get grain alcohol at the liquor store. Is this ok :?: I tried yesterday onion, strawberry, and banana with the blender and it DIDN'T WORK. However I did it by chopping with a knife and it worked perfectly. My hypothesis is if the animal cell has more chromosomes, then you will extract more DNA than a plant will. We are washing everything after each extraction and we have a lot of measuring cups and materials. What do you mean about "contaminating my extracted DNA or interfere with the extraction. " What do you mean about this sentence? "If you break down different percentages of different cell types, then your idea of measuring ratios of extracted DNA to starting cell mass may render any conclusions meaningless." If we use 6 ounces of each food what is the amount of alcohol to pour? Because I think one centimeter is too little.
Thanks Again!
Bryan

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Craig_Bridge
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Postby Craig_Bridge » Fri Oct 19, 2007 3:55 pm

I think that your telling me not to use a blender but chop with a knife, right?
No, I'm was just trying to get you to think things through yourself. If you use a blender to do the chopping, I would recommending you only put in onion (or whatever you are testing) and not add any water and that you measure the mass (weight) of what you take out of the blender instead of what you put into it if knowing how much onion (or whatever) you started with is important to measurements for your hypothesis.
My dad will get grain alcohol at the liquor store. Is this ok
I believe you said you live in Florida. If that is the situation, I don't think you can purchase 190 proof grain alchohol (95% ethanol) for human consumption. Because the alchohol is used in the very last step of this extraction proceedure, it probably doesn't matter if it is ethanol or isopropyl given that people have posted success with both. Using the highest percent alcohol that you can and keeping it cold appear to be more important than ethanol vs isopropyl.
What do you mean about "contaminating my extracted DNA or interfere with the extraction. "
I was trying to inform you that this DNA extraction proceedure might not work very well for things besides onion. Chemical extraction steps are all about either
a) removing the things you don't want and leaving things you do in solution, or
b) precipitating out things you want and discarding the solute. The other cells (stawberry, banana, etc.) may contain chemicals which this particular set of extraction steps may not handle. This set of extraction steps might leave something that binds to the acid end of the DNA molecules and contaminates the DNA you extract. The steps might leave something around that breaks down some of the DNA before it is extracted or might change the solubility of the DNA and precipitate it out early or keep it in solution (interference).
"If you break down different percentages of different cell types, then your idea of measuring ratios of extracted DNA to starting cell mass may render any conclusions meaningless."
Lets do a pretend experiment with pretend results to illustrate: If you mashed up a small piece of banana and pressed it into the bottom of a test tube, then only the top surface of the clump would have cells exposed to the chemicals used to break down the cell walls. The number of cells whose walls broke would be small compared to the total number of banana cells in the tube. If the cell walls don't break, the DNA inside won't end up in the final separation. You could easily end up with less than 1 percent of the banana cell walls broken. This means you could only extract less than 1 percent of the DNA.

Lets pretend that you manage to break down 50% of the onion cells. The results of this unfair experiment are going to tell you that 1g of onion has more DNA than 1g of banana.

Next, pretend that you chop up the onion on a wooden cutting board and you really dice it small and pound it and manage to mechanically break 25% of the cells and the onion juice soaks into the board taking 25% of the DNA with it. Then assume that your extraction process only breaks 30% of the remaining cells, you are going to end up with at most 23% of the original DNA. Compare this with a pretend strawberry sample where you use a mortar and pestle to grind up them up and collect all of the juice and the cells completely disolve in the 100ml of soap water and all of the cells break so you recover >95% of all of the DNA. Is this a fair test?

Enough fiction. What about cell density? Does the average onion cell weigh more or less than the average strawberry or banana cell? You likely aren't going to be starting with the same number of cells and you probably aren't going to be recovering the same percentage of the total DNA in your samples, so what are your results really going to tell you?

Your proceedure for extracting DNA from plant cells probably isn't going to work for animal cells. Here is a college biology lab reference http://biology.arizona.edu/sciconn/lessons2/Vuturo/vuturo/dna.htm for collecting and separating DNA from human cheek cells. The initial steps are quite different. Only the final separation step is similar.
-Craig

bryanandsebastian
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Problem with chicken............

Postby bryanandsebastian » Mon Oct 22, 2007 6:12 pm

Hey Craig,

I'm wondering why these procedures for the onion method won't work with the chicken liver. We haven't done it yet but, we're are trying to do fish eggs, chicken eggs, and chicken liver. Also we would like to try yeast. Again thanks for the help and all your advice. :D
Bryan

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Louise
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Re: Problem with chicken............

Postby Louise » Mon Oct 22, 2007 7:53 pm

bryanandsebastian wrote:Hey Craig,

I'm wondering why these procedures for the onion method won't work with the chicken liver. We haven't done it yet but, we're are trying to do fish eggs, chicken eggs, and chicken liver. Also we would like to try yeast. Again thanks for the help and all your advice. :D


Plants and animals have cells made of different chemicals (as well as different ratios of similar chemicals). For example, one chemical that is found in plants but not animals is cellulose. (This is why wood is so hard; the cell walls have cellulose in them). The goal of this prep is to extract only one type of chemical and discard all the rest. If "all the rest" are different, they will require different protocols, ad Craig found when he researched this topic. The methods for preparing the plant vs. animal cells are very different. Even within plants the method you found may not work for all types of cells.

Does this help?
Louise

Craig_Bridge
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Postby Craig_Bridge » Tue Oct 23, 2007 8:40 am

Louise gave you the short answer. Neither of us are DNA extraction experts but we know just enough to have concerns and suspicions.

If you look at the Lysis Buffer prep in the materials list for the lab http://biology.arizona.edu/sciconn/lessons2/Vuturo/vuturo/dna.htm for extracting DNA from cheek cells you will see it contains:

5 mL 1M Tris pH 8.0
10 mL 0.5M EDTA pH 8.0
5 mL 1M Sucrose
5 mL 10% SDS
2 mL 5M NaCl
Add distilled water to bring volume to 100 mL. Mix well.

This is quite different from what you used in the Lysis step for your onion DNA extraction. In addition, a centrifuge step is needed.

Note: Then there is the mention of Proteinase K which is expensive but can be used to increase the DNA yield of the extraction steps.

Also note: The contents of cheek cells are far less complex than liver cells so this even this Lysis Buffer might not be good enough. Eggs have a whole lot of nutrients in them that neither cheek cells nor liver have so there might be another variation on Lysis chemistry required.

In short, these are concerns.
-Craig

Louise
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Postby Louise » Tue Oct 23, 2007 9:27 am

I couldn't find the lysis buffer for the cheek cells last night, so thanks for the info Craig.

Here is some info on what the different components of the buffer are doing:

Cheek Cell Lysis Buffer
5 mL 1M Tris pH 8.0 - this keeps the solution at proper pH (acidity or basicity). Proteins get messed up at the wrong pH, so almost all biology people work in pH buffers. Your simple onion prep doesn't have this; it may not be important if you don't care about the quality of the DNA.

10 mL 0.5M EDTA pH 8.0- this is a chelator and binds metals ions, of which there are many in cells (both plant and animal)
See: http://en.wikipedia.org/wiki/Tris for information on these first two ingredients.

5 mL 1M Sucrose- This is sugar. I honestly don't know why it is in the prep. I'd guess it was added to maintain a certain oscmotic pressure on the cells, but that is a complete guess. You said your mom worked in a microbiology lab, maybe she can ask someone (or she knows).

5 mL 10% SDS- This is a type of pure detergent. It is probably what is in your dishwashing soap. It has the full name of sodium dodecyl (lauryl) sulfate. It helps break up the cell membrane, which is made of long fatty things called lipids. It will also help remove the proteins that are found in the membrane. The items are not soluble in water, so with out the SDS to coat them, you could not remove them.

2 mL 5M NaCl- Salt. see below...

I'm guessing you are using this prep for the onion:
http://www.funsci.com/fun3_en/dna/dna.htm

Your prep uses heating to remove destroy some of the chemicals (or cause some of the proteins to become broken and fall out of solution. Think of what happens when you curdle milk), and then just uses salt and detergent. You could probably extract DNA from chicken liver with it, _but_ is isn't optimized at all for that type of cell, so you could get a very misleading result (either you might extract other things as well as DNA , or you might extract only a small fraction).

Your prep claims the salt is for -"We will also use a little table salt, which helps to eliminate the proteins, called histones, on which the DNA is wrapped." I guess the hydrogen bonds between the histones and the DNA are disrupted by the salt, but this effect certainly isn't specific to DNA/histones. [ I'm not sure why this histone salt complex would not go to the ethanol layer with the DNA.] Then later, it says to use pinapple to remove more histone, so I guess this isn't very efficient.

Anyway, if your hypothesis is based on the quantity of DNA, then it is probably an unfair (and non-scientific) comparision to look at different cell types. I'd stick to vegetables.

As Craig mentioned, I'm not an expert on this. I work a little with proteins, but I'm answering your questions with my biochemistry book in my hand! We just want you to be aware of certain complicating factors that we see with your experimental plan.


Louise

Angelia9289
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Postby Angelia9289 » Thu Nov 08, 2007 8:03 pm

i need some help i have decided to do this experiment for science fair can someone please tell me what is EDTA? my title of my project is Will DNA survive seperated from a cell component? this has something invoving an onion
Angelia M

Louise
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Postby Louise » Thu Nov 08, 2007 8:22 pm

Angelia9289 wrote:i need some help i have decided to do this experiment for science fair can someone please tell me what is EDTA? my title of my project is Will DNA survive seperated from a cell component? this has something invoving an onion


I don't think you need EDTA for the onion project. You should read the instructions carefully. As for what EDTA is, please read what I provided above and am quoting below again:

10 mL 0.5M EDTA pH 8.0- this is a chelator and binds metals ions, of which there are many in cells (both plant and animal)
See: http://en.wikipedia.org/wiki/Tris for information on these first two ingredients.


This article talks about EDTA. If you want more information, wikipedia also has an article just about EDTA.

Louise

Angelia9289
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Postby Angelia9289 » Thu Nov 08, 2007 8:34 pm

ok thanks i found two different material list for this project i think i will do the one that provides the less complicated one
Angelia M


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