Science Buddies: "Ask an Expert"

So I've run into a little problem...but here's my project:

I'm going to try to find the optimal concentration of SDS (a detergent that is essential to extracting DNA) that will yield the most DNA is a simple collection method. But I don't know if this project seems to obvious, like isn't the obvious answer that the highest concentration of SDS will yield the most DNA? Also, should I use a dry detergent, a liquid detergent, or try to find the pure chemical online?

Another issue, if i use the pure chemical, I may need to hand in extra forms which are due December 1st and have to be approved before I start the project..and it's really more work than its worth. But the other thing is that I may not have to hand in the forms if the chemical isn't toxic..so is it? I can't seem to find any answers! Please help!

Hi Beccan,

This sounds like it is an excellent science fair project idea. Doing a carefully controlled experiment on a basic procedure that many scientists use for their research is definitely a worthwhile idea. Progress in science is made one experiment at a time, so I would encourage you to continue.

The Wikipedia article includes some background information on SDS, and reference 6 is a link to a database for household products that include SDS. Please note that you will have to search using all of the alternative names for SDS, such as sodium lauryl sulfate to find all of the products that contain this detergent.

http://en.wikipedia.org/wiki/Sodium_dodecyl_sulfate

You would need to look up some of these products and try to pick one that does not have any other ingredients. If you can find one that does not contain other ingredients, this would be a perfect choice for your experiment.

However, it might be best to try to purchase the pure reagent grade SDS, even if it means filling out the forms. Using the pure detergent would ensure that you would not be including unknown variables in your experiment with the reagent.

Here is the information from this website about the rules for projects involving hazardous chemicals.

https://www.sciencebuddies.org/science- ... chem.shtml

SDS is a detergent and while it would be a good idea to wear safety glasses and gloves when working with the pure substance, it is not particularly hazardous, so I don’t think you would need special approval for working with this reagent. However, do check with your teacher and go ahead and submit the approval forms if there are any questions about the requirements for approval. Your project could be disqualified at judging if it is entered without required approval.

https://www.sciencebuddies.org/science- ... chem.shtml

Here is an material safety data sheet (MSDS) for SDS that includes precautions for handling this chemical.

http://www.sciencelab.com/msds.php?msdsId=9925002

So what are you planning to do for your experiment? How are your going to measure your results?

Donna Hardy

Thanks! I'll ask my teacher when I go back to school!

I'm going to use different concentrations of SDS or other detergent probably in something like 5, 10, 15, 20, and 25% increments, but I haven't completely decided yet, and see which concentration extracts DNA from strawberries the best. I was planning on measuring the amount of DNA by letting it completely dry and then measuring its weight.

Again, thank you for all your help!

Hi Beccan,

You are welcome!

Your general experimental protocol sounds like a reasonable approach to the problem. What have you learned about this technique from your background reading?

Do you have a spectrophotometer at school that will measure absorbance at 260 nm? DNA absorbs UV light at 260, and proteins, which absorb UV at 280 can also be extracted by SDS. Measuring the weight of the extracted sample will give you a good quantitative measurement, but you would still need a method to verify the purity of the DNA.

Donna

The technique I'm using is the most common protocol for school labs, and it also seemed simple enough to be done several times. Basically it's like the do it yourself DNA on this site.

Is there anyway to test the purity of the DNA without using the spectrophotometer? I don't think our school has one because our science department is kind of lacking in funds... Would the proteins being extracted as well as the DNA count as an experimental error?

One more thing: I'm not sure how to say doceyl...is it like doe-des-il or doe-dek-il?

Hi Beccan,

One issue that you need to address is the purity of the samples, so you do need a method to verify the identity of your sample. The SDS will cause cell lysis to allow release of the cell content; the alcohol step will precipitate large biomolecules, primarily DNA, but it would also be possible to copurify some proteins.

Ask you teacher if any Coomassie brilliant blue or Protein Dye reagent is available. The dye will turn blue when it interacts with proteins, but SDS does interfere with the reaction. Also, ask if there are any DNA-selective dyes available such as Fast Green or ethidium bromide.

Good question. Docecyl is pronounced “doe-des-il” (like the word decimal).

Donna

What would I do if the sample contained proteins as well as DNA? Would I have to scratch the entire thing and start all over until I get one that is pure DNA?

Also, how do I use it?

Hi Beccan,

1. Not necessarily. Your independent variable will be the concentration of SDS you use to lyse the strawberry cells and the quantity of DNA in the sample would be your dependent variable (protein, if present, would be another dependent variable.) If you can confirm, even with a qualitative test that there is no protein, or maybe just a small quantity, then you will be able to support your hypothesis. And it will be a better experiment if you can verify the identity of the DNA in the samples. If you don’t find another method, you can describe the appearance of the sample or take a photograph to verify that the appearance is consistent with DNA.

I had not looked at the procedure for a while, but here is a typical protocol. One of the steps is to add meat tenderizer, which is an enzyme that breaks down proteins. Are you including this step? If so, then it will eliminate the problem of potential protein contaminants, except that papain is a protein itself, so you would need to make sure that you added the same amount to each sample (controlled variable).

http://learn.genetics.utah.edu/content/ ... action.pdf

2. How do you use what? Are you referring to the Coomassie or the DNA stain? Please verify what you have available, and I’ll help with a protocol. Otherwise, I’ll try to think of something else to use.


Donna Hardy

Thank you so much! I had not originally planned to use meat tenderizer, but now I will! I'm still a little confused though, will I still need to confirm/deny the presence if proteins? (I do still plan on asking my teacher if we have either of those tomorrow.)

Dear Beccan15,
I would still confirm the presence of proteins in your sample. As donnahardy2 has mentioned, you can do this by use of a comassie blue stain, but keep in mind that most DNA is wrapped around histone proteins so you should expect a small amount in your sample.(there will generally be some blue staining from the comassie)

Would the experiment be a failure if there are proteins present though? Or could I still do a dry measure and just say when I present that I also extracted proteins as well as DNA?

Hi Beccan,

Your experiment as you have designed it is excellent and you don’t have to do anything else. It’s a perfectly controlled experiment and meets all of the requirements for a science project.

However, as Tyber has suggested, the experiment would be better if can do some additional analysis on your samples to verify they contain pure DNA. Looking ahead to the science fair, the judges will be interested in knowing about this as well. If you can’t get anything else to verify the purity of the samples, you should at least be able to explain what you would have done if it had been possible, jut to show you understand the concept.

Let us know if you can obtain anything that would test for total protein and DNA.

Also, I recommend that you do a pilot experiment and run one test sample as soon as possible, to see if you are going to have any technical difficulties in drying the samples for weighing. You want to solve any procedural problems before you do the definitive experiment with all of the different concentrations of SDS.

Donna Hardy

Thank you all for all your help! My teacher was going to check our school's chemical stocks tonight and tell me tomorrow hopefully, and I'll run a pilot test as soon as all my forms are in! (It's against the rules to do one beforehand)

Hi Baccan,

Great! Let us know what your teacher can find. We can make suggestions based on what is available.

Donna

So my teacher told me that in bio II they use biuret solution a an indicator of proteins, but she doesn't know if it would work for my experiment, so my question is would it?

One more thing, do you think 5-25% in 5% increments is too big a range? Should I maybe use 5-10% in 1% increments instead?

Hi Beccan,

This is good news. The biuret test is a general, all purpose total protein assay, so should work well for your experiment. And, I believe that it is compatible with SDS. You will need to set up a standard curve with a known protein to establish a standard curve, and then compare sample results to the standard curve. Do you have anything available to use as a standard?

If you don’t have a spectrophotometer, you can set up the standard curve and just compare the density of the color in each tube to estimate the protein concentration. The blue color will be darker with higher concentrations of protein.

http://en.wikipedia.org/wiki/Biuret_test

I recommend trying the 5% increments first and then decide if you want to try using other concentrations. If there’s no difference between 10 and 15%, then there’s no point in doing 11 and 12%. Each sample is going to be a lot of work, and you want to allow enough time to do your experiment twice. Or at least do the pilot experiment, and then the definitive experiment in duplicate.

What about DNA stains? Is there anything available that is specific for DNA?

Donna Hardy

From what i read on the biuret test, I'm presuming that I would take the DNA out from the strawberry-alcohol suspension and maybe use a pipette to place a drop of the solution on the sample? Or do you have a better way of doing it?

My teacher is asking other chemistry teachers if we have a DNA stain, but she said that it is likely we don't have one that I would be allowed to use (I don't really know why I wouldn't..)

Hi,

Yes, basically all you have to do is mix the sample with the biuret reagent to develop the color. There are 3 steps to the protocol:

http://www.brilliantbiologystudent.com/biuret_test.html

What is the sensitivity of the biuret test? If you obtain a negative result on this test, you will want to report the results as “less than x mg/gram pr mL.”

Do you have access to pipettes? The best way to do a quantitative assay is to take a specific volume of the total sample and measure the protein so you can calculate the ug or mg of protein per gram of strawberry. This would also allow you to compare results of all of the samples.

The reason your teacher made the comment about the DNA stain, is that one of the most common DNA-specific stains is ethidium bromide. The stain is carcinogenic and therefore dangerous to work with. However, there are a number of
DNA-specific stains, such as Fast Green, Sybr green that are safe to work with.

Donna

The protocol says to add 2cm^3 of liquid food sample, and then the footnote says it could be prepared from solid foods, so would it be good to use the dry DNA sample after I've measured it's weight and re-dissolve it in water to perform the test?

Also, is the biuret solution toxic? Or dangerous to touch?

I don't understand what you mean about specific volume. How would I perform that test?

Thanks for clearing that up about the DNA stains!

One more thing, will it seem as though my experiment is too complicated if I perform all these tests for DNA and proteins? Last year I had points taken off because I "performed unnecessary tests."

Hi Beccan,

You have a good question, so I will explain a little bit about science fair judging Every science fair has guidelines for projects and the science fair judges are given criteria for judging projects. For example, there are points for every section such as the hypothesis, experimental design, and conclusion and points for creativity and scientific thought.

Here is information from this website on evaluating science projects and it includes criteria that can be used for every section. I recommend that you read through this information to understand how science projects are evaluated.

https://www.sciencebuddies.org/science- ... rces.shtml

You should also look up the rules and guidelines for the science fair that you will be entering.

The problem with judging science fair project is that generally all of the projects are very good and there can only be one winner in each category. If there are 25 really good projects and there can only be one winner, then it is necessary to compare the small details of each project and try and pick the best one. Sometimes there is only ½ point difference between the first and second place winner. That’s why it’s necessary to pay attention to small details when planning your project.

Science fair judges have a variety of experience and background, but generally science fair judging is done in teams, with multiple teams evaluating each project. This helps ensure fair judging and the final outcome is a consensus of the opinion of several experienced judges.

For this project, the basic experiment is good; you have designed an experiment that will compare different concentrations of SDS to determine the optimum concentration for purifying DNA. If I were judging this project, after I checked to make sure that you had designed a well-controlled experiment, understood the scientific principle behind your project, and had included all of the necessary sections, the first question I would ask would be about the purity of the sample. If, for example, you obtained a higher weight with 10% SDS compared to 5% SDS, I would want to know if there was any difference other than the quantity. Doing an additional test to verify the identity of the DNA and checking to make sure the sample does not contain a major contaminant (protein) would help validate your results and support your conclusion. The additional testing would complement the main experiment, and would not be considered unnecessary.

I don’t know what happened in the judging for your project last year. But it’s very important to learn from the past and make sure you don’t repeat the same mistake. What was your project last year? What was your experiment, and which testing was considered unnecessary? What project won compared to yours? If you could explain, I might be able to understand what happened and why the one judge made the comment about unnecessary testing.

Does this make sense? Do you have any ideas that would help make your project even better?

Donna Hardy

Hi Beccan,

Here is an answer to your earlier questions.

Carolina biologicals offers laboratory grade SDS and you might be able to order it directly.

http://www.carolina.com/catalog/search- ... SearchForm

The Biuret reagent contains sodium hydroxide, which has a high pH. and it contains copper. Here is the material safety data sheet for this reagent. You will see that the sodium hydroxide is very corrosive and that the copper is toxic. Wearing gloves, protective clothing and safety glasses are essential when working with these reagents. You should plan to do this test at school, not at home

http://www.sciencestuff.com/msds/C1312.html

A cm3 is a milliliter of volume. Do you have pipettes available to measure the sample and the reagents? You need to measure everything very accurately.

Donna Hardy

In the science fair I compete in, there isn't one "winner." It's a little weird, its basically like you're getting a grade that can be either a first, second, third, perfect score prize and then all the first/perfect prizers go on to the state level. Maybe you've heard of it, it's called PJAS.

My project last year was testing whether an acid or an alkali has a greater detrimental affect on the growth of duckweed. The only thing I tested was the pH of the different solutions, so that's the only thing they could have been referring to. The other projects that got higher scores were road salts on plants, effects of sodium and chloride on lettuce seeds, and I think the last one was something with the effect of gravity or different sound waves..I don't really remember.

My school does not have anything that is safe enough for me (as a sophomore, they have guidelines for what grades/science classes can use which chemicals) to test for DNA, but I am allowed to use the Biuret solution for testing for proteins.

Thanks for that! My dad also has connections at work he can use to get me SDS too.

Yes, I do have access to pipettes, and a very wide variety of sizes too.

donnahardy2 wrote:The best way to do a quantitative assay is to take a specific volume of the total sample and measure the protein so you can calculate the ug or mg of protein per gram of strawberry. This would also allow you to compare results of all of the samples.

When you say total sample, do you mean the strawberry itself or the sample of DNA?

Hi Beccan,

Thanks for explaining about the judging on your project. I am not familiar with this type of judging, but it sounds interesting.

From your description of your project, the pH was your independent variable and the growth of the duckweed was your dependent variable. It sounds like it was a very good project. You should have measured the pH of the samples and the growth of the duckweed. You could have measured other parameters such as temperature, nitrogen content, light intensity, dissolved oxygen, etc. to verify that all other conditions were controlled, but it doesn't sound like you did any extra testing.

So I don't understand what the comment about the unnecessary testing would refer too. In my opinion, having measurable results, and doing additional testing to verify results is great when doing science fair projects.

For the biuret assay, you will prepare each sample and take a small amount for protein testing. For example, you might process a strawberry that weighs 20 grams total and end up with a volume of 30 ml of sample. If you use a 0.1 ml aliquot of the sample for the protein testing, you would measure the protein concentration in the 0.1 ml (100 uL), and multiply by 300 (to obtain the total protein content in the strawberry) and then divide by 20 (to calculate the amount of protein per gram of strawberry).

You will purify all of the DNA from the strawberry and weigh it and divide by 10 to obtain the weight of DNA/gram. In your results section, you can report all results as weight per gram, so you will be able to compare results from sample to sample. Does this make more sense now? Can you write a detailed protocol?

For the DNA verification, you can explain on your project board what you would have done (using ethidium bromide) to confirm the sample contained DNA, but you will state that you didn't actually do the testing because you were not allowed too. Whoever is judging the project should give you extra credit for thinking about doing it.

Donna

Yes, thank you. I was originally planning in using 3 or 4 strawberries at a time and the dividing the blended strawberry mixture in however many containers of 20ml it would fill. Would this work too if I take the average weight of the strawberries or just use the total mass of the group?

Hi Beccan

Preparing a big batch of blended strawberries and dividing it to use for each sample would work well. If you measure the weight of the strawberries before you start, and then measure the weight of the individual 20 ml samples, you will know how many grams of strawberries are in each sample.

You should do a pilot experiment to make sure your experiment will work and that you will have enough sample to obtain a measurable amount of DNA.

Donna

Why do I have to measure the weight of the individual samples?

Hi Beccan,

Because blended strawberries are very viscous and you are going to lose some sample in the blender and it will be difficult to evenly distribute the sample into the individual containers. You don't have to weigh the individual samples, however, it would be better to do so. If you obtain a different quantity of DNA in the samples, it will be helpful to verify that you started with the same amount of starting material.

Donna