DNA collection

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DNA collection

Postby beccan15 » Sat Nov 10, 2012 10:04 am

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!
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Re: DNA collection

Postby donnahardy2 » Sun Nov 11, 2012 10:44 am

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.

http://www.sciencebuddies.org/science-f ... 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.

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

Postby beccan15 » Sun Nov 11, 2012 4:55 pm

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!
beccan15
 
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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 11, 2012 6:45 pm

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
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Re: DNA collection

Postby beccan15 » Sun Nov 11, 2012 8:33 pm

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?
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Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
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Project Status: I am conducting my research

Re: DNA collection

Postby donnahardy2 » Mon Nov 12, 2012 1:40 pm

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
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Re: DNA collection

Postby beccan15 » Mon Nov 12, 2012 3:10 pm

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?
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Project Question: electrophoresis with different plants/fruits/vegetables, haven't decided yet
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Project Status: I am conducting my research

Re: DNA collection

Postby beccan15 » Mon Nov 12, 2012 4:50 pm

Also, how do I use it?
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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 12, 2012 6:34 pm

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
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Re: DNA collection

Postby beccan15 » Mon Nov 12, 2012 8:28 pm

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.)
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 tyber » Tue Nov 13, 2012 8:43 am

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)
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Re: DNA collection

Postby beccan15 » Tue Nov 13, 2012 9:19 am

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?
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 13, 2012 3:20 pm

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
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Re: DNA collection

Postby beccan15 » Tue Nov 13, 2012 4:31 pm

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)
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 13, 2012 5:03 pm

Hi Baccan,

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

Donna
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