DNA collection

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Re: DNA collection

Postby beccan15 » Wed Nov 14, 2012 10:47 am

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?
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Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
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Re: DNA collection

Postby beccan15 » Wed Nov 14, 2012 3:15 pm

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?
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Re: DNA collection

Postby donnahardy2 » Wed Nov 14, 2012 3:35 pm

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
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Re: DNA collection

Postby beccan15 » Wed Nov 14, 2012 7:09 pm

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..)
beccan15
 
Posts: 27
Joined: Tue Oct 09, 2012 4:34 pm
Occupation: Student: 10th grade
Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
Project Due Date: February, Early March 2013
Project Status: I am conducting my research

Re: DNA collection

Postby donnahardy2 » Thu Nov 15, 2012 8:12 am

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
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Re: DNA collection

Postby beccan15 » Thu Nov 15, 2012 9:36 am

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!
beccan15
 
Posts: 27
Joined: Tue Oct 09, 2012 4:34 pm
Occupation: Student: 10th grade
Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
Project Due Date: February, Early March 2013
Project Status: I am conducting my research

Re: DNA collection

Postby beccan15 » Sat Nov 17, 2012 8:42 am

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."
beccan15
 
Posts: 27
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Occupation: Student: 10th grade
Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
Project Due Date: February, Early March 2013
Project Status: I am conducting my research

Re: DNA collection

Postby donnahardy2 » Sun Nov 18, 2012 8:59 am

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.

http://www.sciencebuddies.org/science-f ... 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
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Re: DNA collection

Postby donnahardy2 » Sun Nov 18, 2012 9:12 am

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
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Re: DNA collection

Postby beccan15 » Sun Nov 18, 2012 11:34 am

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?
beccan15
 
Posts: 27
Joined: Tue Oct 09, 2012 4:34 pm
Occupation: Student: 10th grade
Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
Project Due Date: February, Early March 2013
Project Status: I am conducting my research

Re: DNA collection

Postby donnahardy2 » Mon Nov 19, 2012 6:26 pm

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
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Re: DNA collection

Postby beccan15 » Mon Nov 19, 2012 6:43 pm

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?
beccan15
 
Posts: 27
Joined: Tue Oct 09, 2012 4:34 pm
Occupation: Student: 10th grade
Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
Project Due Date: February, Early March 2013
Project Status: I am conducting my research

Re: DNA collection

Postby donnahardy2 » Tue Nov 20, 2012 7:26 am

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
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Re: DNA collection

Postby beccan15 » Tue Nov 20, 2012 9:00 am

Why do I have to measure the weight of the individual samples?
beccan15
 
Posts: 27
Joined: Tue Oct 09, 2012 4:34 pm
Occupation: Student: 10th grade
Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
Project Due Date: February, Early March 2013
Project Status: I am conducting my research

Re: DNA collection

Postby donnahardy2 » Tue Nov 20, 2012 9:15 am

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
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