Extracting Onion DNA

[bryanandsebastian]

Please, I need to verify if the variable in this experiment is the onion and if the controls are the detergent, salt, and alcohol. I also would like to know if I can experment with other fruits or food in this project, because I need data for comparison in order to make graphs or tables which i need for, to make this project. Please answer as soon as possible. I will really appreciate it.

Sincerly,
Bryan

[MelissaB]

Hi,

Our variable guide here: https://www.sciencebuddies.org/mentorin ... bles.shtml should help you determine what a variable is and what variables you have in your experiment.

In the introduction to the project online, it says that they use an onion because of its low starch content. Thus, you might want to do some research and find out what other fruits or vegetables have low starch content and experiment on those.

[bryanandsebastian]

Um.. I'm trying to see what is the control and variable for the experiment in biotechonolgy "Extracting Onion DNA."

-Bryan

[geoffbruton]

Hi Bryan,

Did you go and read the information contained in the Science Buddies Project Variables website that Melissa recommended?

All of the information you need in order to answer your question is right there!

Please either read, or go back and re-read, the information provided. Then, try and reason out which variable is which in your project. If there is something you do not understand, please let us know.

Please post back to the forum with your thoughts, and we'll let you know how you've done.

Good luck!
Geoff.

[bryanandsebastian]

My problem is that this project doesn't really give good data to measure ( or at least for the science fair ) so what I decided is, make the suggestion that the project "Do It Yourself DNA" gives the idea of measuring DNA from other products. This is what I want to do. So this is my conclusion:
The dependent variable is the DNA. The independent variable is the different vegetables and fruits, and lats but not least the controlled variables are the amount of salt, detergent, alcohol, weight of the products, and the temperature.

(P.S. I have my teacher's permission to do this)
Thanks for the advice, please answer back ASAP,
- Bryan

[geoffbruton]

Hi Bryan,

You've done a great job in determining the variables in your experiment!

Now you need to decide which fruits or vegetables you would like to analyze for your project. You'll notice that the project outline in the Science Buddies Project Ideas explains that strawberries are octoploid - meaning that they have eight copies of their DNA in every cell. Since you are planning on intercomparing different fruits or vegetables, you should probably look into how this might affect your yield, and why.

Also, what mass of fruit and vegetables are you planning on analyzing? You correctly stated that this is a controlled variable, but you will need to determine the masses beforehand and make sure that you use the same amount for each test. If possible, I would also recommend performing multiple tests on the same fruit / vegetable in order to see if there is any variation from run to run.

It looks like you're off to a great start, so please just let us know if there are any specific questions you may have.

Good luck!
Geoff.

[bryanandsebastian]

Thank you very much for the answer! I need to get this information: how many chromosomes does kiwi, green peas, and chicken liver, have? It's ok to stain chromosomes with methylene blue, or can you recommend any other stain? What are the products or food that yield more DNA? By the way I'm in seventh grade. Thank you again for all the answers Geoff.

-Bryan

[geoffbruton]

Hello again, Bryan,

It sounds as though your project is definitely shaping up! Great work!

Okay, I had a go at trying to find the answers to your questions, and although it took a bit of work (and I certainly didn't know the answers!), the answers are out there! Just as before, go ahead and try typing your question into your favorite search engine and see what you find.

I used Answers.com and Google.com for each of your possible variables and didn't have to go very far. Try varying your keywords and see what you get back...

The Genome News Network (GNN at http://www.genomenewsnetwork.org/resour ... _2_1.shtml) is very helpful with lots of great information.

In addition, the National Center for Biotechnology Information (NCBI at http://www.ncbi.nlm.nih.gov/mapview/) also has a lot of excellent information.

Please do not worry if the vast majority of this information doesn't make any sense - the websites are not aimed at science fair project students! However, they are a great - and scientifically valid - source for the information you are seeking.

I'm afraid that this subject is not really my area, so hopefully another expert who actually works in this field will be able to answer your question as to which is the better stain to use. Could you also please expand upon what you are intending to do with the stain, since this was not in the original experiment.

As for your last question - which foods or products yield the greatest amount of DNA - that is the objective of your project!

Best of luck with your research, and please be sure to post back with how your project is going.

Good luck!
Geoff.

[bryanandsebastian]

Thank you so so much for the info. Apparently all the websites you gave us ( thought we have not checked yet ) looks to me we haven't found those. I don't know how you do it but your doing a great job at getting info. By the way it must be so hard work to take your time and search for our answers! Regarding the stain, my mom works in a lab as a microbiologist and she's gonna try to stain the muccus that contains the DNA. But it's ok she will try to find the answer her self. I'm gonna put a site for you to see: http://www.funsci.com/fun3_en/dna/dna.htm

THANKS FOR THE INFO,
-Bryan[/b]

[geoffbruton]

Hi Bryan,

Glad to be of help! But please don't misunderstand - I'm not *giving* you the answers, I'm just seeing if *I* can find them, and then simply showing you that the information is out there. You are the one doing all of the real work - and doing a great job of it!! Like so many of the Science Buddies Experts, my work often involves researching different subjects - so we often get pretty good at finding things quickly.

Thanks also for the website you provided - I thought it was excellent and I hadn't found that one! A great find with some terrific photographs - which makes me think that perhaps you should try taking some photographs of your work and including them in your presentation.

You are also very lucky that your mom works as a microbiologist - please let us know what you both decide would be the best way to go with the staining part of your project.

Keep up the good work, and please keep us informed of your progress.

Good luck!
Geoff.

[bryanandsebastian]

Hi again it's Bryan!

I need to make sure which onion (white or yellow) is better to do the experiment. Another Q. is if I can use 95% Isocropyl Alcohol instead of 95% Ethanol. Please answer as soon ASAP because we are going to start my project tomorrow ( 10/13/07 ).

-Bryan

[geoffbruton]

Hi Bryan,

Glad to hear that you're almost ready to start your experimental phase!

With regards to which type of onion to use (yellow, white or even red or green), I think that is entirely up to you. I have no idea which might contain a greater or lesser amount of recoverable DNA - though this could always be something that you could look into as part of your project! As long as you specifically state *which* onion you analyzed, I think you'll be safe.

As for which alcohol to use - do you mean isopropyl alcohol? (I'm not sure what you mean by "isocropyl" - do you have any more information on this material?) To be honest, I have no idea as to whether or not this will have effect on your results - hopefully someone else in the field will be able to give you a better idea. Is there any reason why you do not / cannot use the 95 % ethanol used in the original paper? This should be readily available to you through your school.

Please let us know your thoughts - and good luck!
Geoff.

[deleted-71588]

Isopropyl alcohol C3H7OH and ethanol alcohol C2H5OH are both hydrocarbon solvents with an OH ion; however, that is about the end of their similarities in chemical reactions because they are very different molecules.

Without reasearching your process from beginning to end, one can't answer if one can utilize a different alcohol solvent. In other words, you would have to do a considerable amount of experimentation to determine if it makes a difference or not. Since the difference in various alcohol solvents in a DNA extraction isn't your experiment, stay with ethanol.

[bryanandsebastian]

We have no 95% ethanol in the stores to use. They don't sell it commercialy. We've seen in other research that they use 91% isopropyl
We've found that the difference is that there is more water in this one. By the way what's the control in this experiment. The teacher says it should be a known source of DNA to compare to our results. How do we know that what we are extracting is real DNA? We know that the answer is that the muccus we get is the DNA, but she keeps asking us for the control. For example in "which is the best mouthwash" the control is distilled water. Or the control is the experiment itself???

Thanks,
Bryan and Sebastian

[Louise]

We have no 95% ethanol in the stores to use. They don't sell it commercialy. We've seen in other research that they use 91% isopropyl
We've found that the difference is that there is more water in this one. By the way what's the control in this experiment. The teacher says it should be a known source of DNA to compare to our results. How do we know that what we are extracting is real DNA? We know that the answer is that the muccus we get is the DNA, but she keeps asking us for the control. For example in "which is the best mouthwash" the control is distilled water. Or the control is the experiment itself???

Thanks,
Bryan and Sebastian

Hi! In the USA there is a brand of alcohol called "Everclear" which is available as 95% alcohol (190 proof) and 75.5% (151 proof). In some states the 95% cannot be sold, but most states it can. I would see if your parents can buy this at the liquor store. I think the majority of the sales of this brand is to scientists who need 95% ethanol for experiments. If you are in another country, there is probably something similar.

If you are diluting the ethanol with water, the lower proof may be okay (use less water... post the procedure and someone can help with the math).

I'll let an expert who has been following your thread more carefully answer your question about controls.


Louise

[deleted-71588]

I've done some searching and found http://www.usc.edu/CSSF/History/2004/Projects/J0404.pdf and http://www.geocities.com/CapeCanaveral/ ... -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 https://www.sciencebuddies.org/mentorin ... thod.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.

[bryanandsebastian]

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

[deleted-71588]

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.

[bryanandsebastian]

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 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!

[deleted-71588]

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/less ... ro/dna.htm for collecting and separating DNA from human cheek cells. The initial steps are quite different. Only the final separation step is similar.

[bryanandsebastian]

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.

[Louise]

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.

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

[deleted-71588]

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/less ... ro/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.

[Louise]

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]

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

[Louise]

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]

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