Science Buddies: "Ask an Expert"

Hello
I have a few questions about my science fair!
Okay, I was thinking about a project: which household detergent finds the most amount of DNA in different kinds of fruits like apples, pears, kiwis, etc.
I was going to also find out if different amount of the detergents will change the outcome of the amount of DNA?
and I thought this was a good project-i am in the 11th grade! And, I found it online and it said: 10-12th grade. But the only problem is that I found a similar project on science buddies: finding the DNA in strawberries.
This was a grade level of 4th-5th graders. This is ridiculous.
So I want to know which is right? Is this an okay project for a 11 grader?
PLEASE REPLY SOON! THANK you for your time and patience!

The reason the Science Buddies project is for younger students is because it is not a quantitative project (i.e. they are not measuring anything, they are just extracting the DNA, which is relatively simple). In your project, it sounds like you would actually be measuring how much DNA you get in the end after changing a piece of the experiment, which is much more complicated. Your project topic is fine and is at a high school level. I hope you use our experiment as a guide. Another idea would be to change the concentration of salt added and see what that does.

There are a few ways you could measure the concentration of DNA.

Simple method:
Take ONLY the precipitated DNA from the test tube and let it dry. You can then weigh the dry weight.

More complex:
You could use a UV spectrophotometer to measure the concentration. Your school might have one. Here is a website that explains this: http://people.hofstra.edu/beverly_clend ... NA&DNA.htm

If you have any other questions let us know.

Thank you so much for the reply!
I will do this project and your idea of an UV Spectrometer has also had me thinking. I think I will use that device to find the concentration of the DNA? Am I right?
I have a few questions though, when it says "concentration", why do we need to find that? Is that relevant to my project?
Also, will use the Introduction page and the procedure it gives in order to use the UV Spectrometer? Am I suppose to follow those exact instructions?
Also when it says: "Be sure to remove all of the ETOH after the wash. Place 100µl sample of your blank (nH2O, TE, whatever your DNA or RNA sample is dissolved in) in the cuvette chamber."
I am a bit confused about what "blank" is? It says, nH20 AND TE but I am not sure what they mean?
THANK YOU SO MUCH FOR YOUR TIMEEE!!!!!!!!!!!

heybah wrote:Thank you so much for the reply!
I will do this project and your idea of an UV Spectrometer has also had me thinking. I think I will use that device to find the concentration of the DNA? Am I right?
I have a few questions though, when it says "concentration", why do we need to find that? Is that relevant to my project?
Also, will use the Introduction page and the procedure it gives in order to use the UV Spectrometer? Am I suppose to follow those exact instructions?
Also when it says: "Be sure to remove all of the ETOH after the wash. Place 100µl sample of your blank (nH2O, TE, whatever your DNA or RNA sample is dissolved in) in the cuvette chamber."
I am a bit confused about what "blank" is? It says, nH20 AND TE but I am not sure what they mean?
THANK YOU SO MUCH FOR YOUR TIMEEE!!!!!!!!!!!

Yes, you are right about using the spectrophotometer to find the concentration of the DNA. You said in your original question that you wanted to see if the amount of DNA would change. Concentration is one way way to measure the amount of DNA that is in solution (it is the amount of DNA in a certain volume of solution). If you want to keep it in solution, you could use a UV spectrophotometer to measure the amount, which would be an advanced method. An easier way, as I mentioned earlier, would be to let the collected DNA dry after it precipitates out and then measure the weight.

If you look at the procedure for extracting the DNA, you end up using alcohol to help the DNA become visible to you by forcing it to become solid (precipitate out of the solution). Theoretically, if you change the amount of one of the materials in the procedure (whether it is salt or detergent, you could pick one of them or test both --one at a time), the amount of DNA that precipitates out will be different.

In order to isolate the DNA, I would follow the procedure of one of our projects (either the onion or strawberry DNA one), EXACTLY. For the UV spectrophotometer, there are probably many other online sources that have similar methods, if that one is confusing to you. You should follow their procedure as close as you can (though it may vary slightly depending on the brand of spectrophotometer you use). The important part would be to learn how the spectrophotometer works. You can use the wavelengths of light they give you to determine the concentration. In very basic terms, a spectrophotometer shoots a certain wavelength of light at the solution and it detects what is absorbed by the solution. Different molecules absorb different wavelengths of light more efficiently, so you actually see a peak in the data where it absorbs better at that wavelength. You can use this peak to determine what the concentration is (if the peak is higher, the concentration is generally higher). I know a lot of that webpage I gave you might sound really confusing, but try to read as much as possible about light in general, the Beer Lambert Law, spectrophotometers, ask your teacher, and eventually it will make sense.

As for the "blank" they mention, you need to use the exact same solvents (liquids) that the DNA is being stored in, and will be analyzed in the sample. "TE" is a buffer, and nH2O is (I think) basic water, though it may be purified in some way or bound to another compound. (You can read more about TE here: http://en.wikipedia.org/wiki/TE_buffer). Both of those would be used to keep the DNA "fresh" so to speak, so it doesn't degrade (if it degrades, it would become a different compound and your data will get messed up). So if you used only TE, you'd want to use TE as the blank. If you used a combination of nH2O and TE, you would want your blank to contain the exact same proportion of those (I'm not actually sure the best way to store DNA, I haven't worked with it, so you should do some research on which solvent/solution would be best). The blank helps to calibrate the spectrophotometer. You can look at it this way: if you used TE as the solvent to store the DNA in, TE is also going to absorb light that the spectrophotometer "shoots" at it. You only want to look at the absorption by the DNA, NOT the TE. What you do when you put a blank in the machine is that you tell it to ignore the TE. It basically will subtract out the TE's spectrum from the total spectrum (DNA + TE), to leave you with just the DNA's spectrum when you analyze the actual sample.

Another thing to keep in mind: generally, you want what you are analyzing to be dissolved before you put it in the spectrophotometer. So I think you would need to redissolve the DNA once you get it out of the fruit or vegetable (ask someone who is a spectrophotometer expert before you do the experiment on the best way to do this).

I hope that wasn't too confusing. Let us know if you have any other questions!

Hello Thank you for your help!!!
But in my variables, i don't actually have a control? What would my control be? I don't have a control because I tend to change the amount of detergents I use and different detergents? so would temperature be a control? but is that irrelevant with my project?
Thank you!

Read more information about variables here: https://www.sciencebuddies.org/science- ... bles.shtml

The controlled variables are the ones that stay the same for the experiment, so yes, temperature would be one (unless you want to test the effect of temperature). Remember that you should only change one independent variable at a time and see how it affects your dependent variable(s).

If for example, you wanted to see how the amount of detergent and temperature affects the amount of DNA that precipitates out of solution, you wouldn't change both at once. For the first set of experiments, you would change concentration each time and keep all other variables (including temperature) constant. For the next set of experiments, you would change temperature but keep the concentration constant, as well as other variables.

In general, you want to do the exact same procedure each time you do the experiment, except for changing your independent variable. So that means keeping everything else as constant/the same as possible.

Oh okay..
so for example, I am testing to find out if the amount of detergent and the different fruits affect the amount of DNA.
So, I would have bananas with 5mL, 10mL, 15mL, and 20mL of detergent and then for kiwis with 5mL, 10mL..ETC.
Would that be fine?
So would my control still be temperature even though I am not actually testing temperature in the experiment?

Also, this is an irrelevant question but I want to block everyone from seeing the posts I make,
so is there a way for me to protect it so you and I are the only ones who can see my posts?

Hi Heybah - It looks like Amber is giving you excellent help and advice regarding your project. The Ask an Expert forums, however, are not private. We can't close threads so that only a student and an expert see certain posts.

I hope you continue to post your questions here. I think you'll find the team of volunteer Experts a valuable resource as you continue your research.

Good luck with your project!

Amy
Science Buddies

You are correct about the detergent amounts and the different fruits.

Just remember that the controls are the things that stay the same (at least as much as you can make them). Since you are not testing whether temperature is going to affect the amount of DNA that comes out, it is NOT an independent variable, so it must be a control.

Think of it like this:

Controls are the things you are trying to keep constant. If you don't want to test how temperature affects the results, then it needs to stay the same across the experiments. It would be like randomly adding a strawberry to the mix when you are supposed to be testing a banana.

If you change more than one thing at a time, you won't know what actually produced the results! Take a look at that link I posted last time about variables. It has multiple examples to look at, too.

As Amy said, there isn't a way to make the conversations private. In fact, even if we could, doing so would prevent other experts from helping you (more brains are better than one)! As long as you don't post any personal information like your name, email address, or phone number (which is normally taken out of the message by a moderator if they see it), you don't need to worry. You are anonymous here. Science Buddies takes internet safety very seriously, as you can see here: https://www.sciencebuddies.org/science- ... rnetsafety

Okay thank you so much!!!
You have helped so much with my experiment!
One more thing, in my experiment I am going to take your idea of using a spectrophotometer instead of finding the weight of the dry DNA. So, does that mean I don't have to add ethanol or alcohol to my experiment which means that I don't have to include ethanol in my procedure at all? THANK YOU both of you for helping me and letting me know about the Privacy System of Science Buddies

I am planning to use the fruits: mangoes, kiwis, nectarines? or pears? or peaches? What kind of fruits would be better to find the DNA?

Although you won't be using the dry weight, you still need a way to purify the DNA and separate it from the rest of the solution. So you would still need the alcohol in order to do that. You would then have to dissolve the DNA in an appropriate solution (make sure to do that gently, as you don't want to destroy the fragile DNA).

I have not done this procedure before, but the instructions on one of our projects (the onion one) said this: "It is both interesting and important to understand the reason for some of the steps in the procedure below. An onion is used because it has a low starch content, which allows the DNA to be seen clearly. The salt shields the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to break down by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution."

So you would want fruit with low starch content. I'd start with onions and strawberries, and see how that goes. Then if you have time to do more experiments, you can add other low starch fruits/vegetables. I think you should start simple for now with the number of fruits you test, and make your procedure more complex (so testing different detergents and amounts of detergent would be good, you could also consider the amount of salt added). Troubleshooting the procedure might take a while, so keep that in mind.

I would pick detergents that use different kinds of enzymes or different amounts of enzymes. I'm not sure if they list that on the bottle though, so you'd have to do some research on that.

So after I filter the solution, I have the DNA amount left over. Do I add ethanol after that?
So I would add ethanol to that solution and then the white mucus would appear? After that, do I use an eye dropper to obtain the white mucus in another test tube and use a uv spect. for that solution?
Does that make sense? Sorry if it doesn't though.
But yes, I think I will also add the salt content and its effect on the DNA content in the experiment. Thank you!

For my fruits, I searched online and I found that Kiwis, watermelon, and papayas have medium/low starch content!

Yes, you would add ethanol after you filter.

For the full procedure, check out this project here if you have not yet done so: https://www.sciencebuddies.org/science- ... p001.shtml

I would copy that procedure exactly for the other vegetables/fruits you try.

You could carefully use an eyedropper to get an alcohol/DNA mixture, and that could be put in a vial to store the DNA. But you would have to get the DNA out and put it in a solvent that will actually dissolve DNA for the UV spec part. You don't want to use alcohol for the UV spectrometer analysis, as the DNA must be dissolved for that to work properly.

Thank you so much for everything. I have one question though. What is the pH level of Palmolive?

Hi,

The link here: http://www.biosci.ohio-state.edu/safety ... LIQUID.htm seems to have a reasonable MSDS for Palmolive 46400 DISHWASHING LIQUID, but the pH may vary depending on the specific product that you are referring to.

The Palmolive website can be found here: http://www.colgate.com/app/Palmolive/US ... ients.cwsp, and on their website, they provide a number for you to contact if you wish to learn more about a specific product (1-800-338-8388).

Alternately, you can directly test the pH of the specific Palmolive product that you are using. Your school will probably have pH paper or an electronic pH detector.

Hope this helps!
Aaron Lin

Thank you so much! It gives the pH level Thank you for your help!

Edit: I didn't see that there were two pages of posts. Thanks for helping Aaron!

Sorry it has taken so long to get back to you, I've been on vacation. I'm not sure what the pH of palmolive is. I tried looking at wikipedia but they don't have an article on it right now. If you buy some pH paper strips, you could very easily get a rough idea of the pH. You can order pH paper here: http://www.hometrainingtools.com/ph-pap ... H-PHSTRIP/

Good luck!