Will DNA survive when seperated from other cell components

[charlescullen28]

Hi... I'm doing a science fair project on separating DNA... I will be using an onion for this experiment. I have a question.... I need to know why people want to separate DNA and what are the benefits in doing so.... and also, are there other things i can experiment on besides an onion..... Also some people wondered if it was safe to separate DNA.. I would need an answer to that... I already know that DNA can live by itself for man many years, but i just needed a subject to do and I chose this one.. So that's what I'm doing, oh and if there's more advice you can give or any other help than please feel free to send me advice or opinions.... Thanks for listening

[Walker]

Hi there,

Purification of DNA from plant or animal tissue is a basic lab technique that is a starting point for all kinds of experimental procedures in molecular biology labs. The answer to the question "why would you want to purify DNA?" will vary depending on who you ask! In my case, as a geneticist working with mice as a model system for understanding neurodegenerative diseases, I need to be able to purify small amounts of DNA from mouse tissue in order to run further tests on the DNA so I know which mice have the genetic modifications I want to study (for example, certain combinations of artificially produced "knock-out" alleles). A friend of mine is interested in understanding DNA repair processes, so she isolates DNA from cell culture experiments so she can run tests on it to measure DNA fragmentation. Another friend is interested in how chemical modifications of DNA that are passed on to offspring affect their behavior, so she isolates DNA from mouse brains to scan it for certain kinds of these "epigenetic" modifications. Suffice it to say that there are about as many different reasons to need to isolate DNA as there are experimental protocols.

As far as your choice of tissues is concerned, you can get DNA out of pretty much any living tissue. For your purposes, the easiest tissues to use will be ones that are cell-rich (so not tissues that are predominantly extracellular matrix, such as cartilage, a crustacean's exoskeleton, or wood) and not too hard to blend up (so not, for example, bone). Meat or pulpy (not too fibrous or woody) plant tissues are pretty easy.

DNA isolation is not a dangerous procedure as long as you use good laboratory safety practices and the appropriate personal safety equipment. Depending on the protocol that you use, some of the reagents used can cause burns if they come in contact with sensitive tissues (phenol and sodium hydroxide, for example, are often used in DNA isolation procedures and can cause chemical burns of skin or eyes). Standard laboratory safety precautions (wearing laboratory safety goggles, latex gloves, and a lab coat) are sufficient to make DNA isolation quite safe. (Phenol is really about the nastiest chemical you'll encounter in most lab's DNA extraction protocols so you may want to avoid using it for your project; it also requires special disposal precautions that may be inconvenient for you to follow.)

You can find many protocols for isolating DNA from various kinds of tissue by googling "DNA purification protocol" or "DNA isolation protocol." Here's a sample protocol that would work fine for a variety of DNA sources:

http://www.molecularstation.com/dna/gen ... isolation/

Here's a "virtual lab experiment" you might find helpful in understanding the steps of DNA extraction:

http://learn.genetics.utah.edu/content/labs/extraction/

Once the DNA is separated from the other components of the cell it's pretty stable. There are DNA-degrading enzymes present in any tissue lysate that will degrade DNA over time if you don't purify the DNA away from them. If you are interested in studying this step in the degradation of DNA you can purchase DNA-degrading enzymes (DNAses) commercially (As enzymes go they're pretty inexpensive, something like $70) or rely on their ubiquitous presence in tissue samples and explore their conditions of action. There are also various chemicals and conditions that can impair the activity of DNases (Salt conditions, pH, temperature, dehydration, etc.)

Good luck!

-Will

[charlescullen28]

Thank you so much, that really helped..... I also have another question.... when i collect data how should i draw my graph since im trying to find out how long DNA will survive... Technically I have a problem in drawing my graph and im not really sure on how to do it....... Oh and how can I tell if the DNA is alive? Wait let me rephrase that........ HOw can I tell if the DNA survived, because i will be using a stopwatch to time it, but how can I tell if it survived or not........

[Walker]

The best way to present your data will depend on the kind of data that you collect.

It sounds like your next task is to figure out what assay you can perform to determine how much DNA degradation has taken place in your experimental samples. This will depend very much on the kind of equipment you have access to.

In a molecular biology lab there are several different common ways of "looking at DNA," any of which could be the basis of a workable assay for you if you have access to the appropriate equipment. The simplest way is based on the light-absorbing characteristics of DNA. A purified solution of DNA absorbs light with a peak absorbance around 260 nm (this is the wavelength of light that is most efficiently absorbed by DNA, in the ultraviolet range), so you can estimate the concentration of DNA in solution using a pretty basic lab spectrophotometer.

Another common way of looking at DNA degradation is to perform electrophoresis on the DNA sample, separating it out by size in an agarose gel and then staining it with a dye called ethidium bromide so you can see the DNA if you shine a UV light on it. If DNA degradation has occurred, you'll see a shift of signal from high in the gel to low in the gel, as small fragments of DNA travel faster and farther during electrophoresis. (There's an animated explanation of this technique at
http://learn.genetics.utah.edu/content/labs/gel/ )
This is a very basic procedure in molecular biology labs but it is more equipment-intensive than a spectrophotometric assay, as you need an electrophoresis setup and a UV light. If you have access to the right equipment it is quite straightforward. If you don't have access to a lab with electrophoretic equipment but you are handy with tools, it is probably possible to make your own electrophoresis equipment (if you're sufficiently motivated!), although this is a very "old school" activity that I have never done myself!

More complex approaches could include quantitative PCR (qPCR) to detect changes in copy number of a particular DNA sequence within the genome. (Here's an intro to PCR from the Utah site:
http://learn.genetics.utah.edu/content/labs/pcr/
qPCR is basically just PCR that is done in a special machine that lets you track how quickly product is produced, which lets you quantify how much of the target DNA sequence was present in the starting sample.)

Why don't you post back with info on what kind of equipment you have access to for this project, as that is the major consideration in figuring out which assay you should use. Once you figure out how you are going to measure the degradation of DNA, you will be able to start thinking about the kinds of questions you can ask about DNA degradation using your assay.

Best,

Will

[charlescullen28]

yeah............ our school has a lab, but I'm not sure if it has any stae of the art stuff I'll ask my teacher if our lab has any of those items..... we mainly only have the basic light microscope and a lot of chemicals, I'm pretty sure that we have the right chemicals but about the equipement? Lets just say that the school lab isnt worthy enough to be a lab...... its more like huge kitchen with a lot of sinks in it and a bunch of chemicals, dangerous and non, but not that much stuff........i'm pretty sure that we have somethings.... When i have the information i'll pass it back on to you.... and thanks for the help

[charlescullen28]

He he........... our lab does not have any of those equipments... um is there a way i can make my own equipment.........

[Walker]

The Science Buddies website actually has a sample project that describes how to make a simple gel electrophoresis setup that might work for you. Take a look at

https://www.sciencebuddies.org/science- ... p028.shtml

There's also a guide to building a gel electrophoresis chamber at
http://learn.genetics.utah.edu/content/ ... elchamber/

While the how-to on the ScienceBuddies site calls for the use of 9 volt batteries to power the elecrophoresis, you can get faster results with a simple DC power supply you can build yourself. An example of how to build a power supply for electrophoresis is at

http://www.science-projects.com/PowerSupply.htm

You could use this apparatus to separate DNA out in agarose gels and visualize the DNA using a chemical stain. (You'd need to purchase agarose and the DNA stain) You could see by the strength of the staining and the relative size of the DNA pieces in the gel how much DNA degradation had occurred in your samples.

I also came across a nice simple protocol for DNA extraction using commonly available supplies here:
http://learn.genetics.utah.edu/content/ ... ion/howto/

If you get a homebuilt electophoresis setup working for you, you'll have the basic tool to visualize the DNA of your choice in a gel. You'll just have to decide what question you want to ask about DNA degradation using it!

Best,

Will

[charlescullen28]

ok and also i asked my science teacher if we could get some of those things and he told me the school ordered some of those things..... but im still planning to build for myself..

[charlescullen28]

ummm i have another question..... What happens to extracted DNA under heat...

[deleted-71827]

Hi,
Extracted DNA under heat will degrade. A further explanation, if you are interested, can be found at this website:

http://www.newton.dep.anl.gov/askasci/m ... e00136.htm

Best of luck!

[charlescullen28]

I also have another question.... That link was extremely helpful in clearing up some confusion I had,......... But after I experiment and start to collect data.... How should I collect it...... should i use a graph or a table? What i'm experimenting is extracting DNA letting it sit for a couple minutes. then run it through an electrophoresis. the second time I extract DNA... I'll put it under heat for a couple minutes......... then run it through the electrophoresis again.... But I'm still not sure on how to collect the data

[deleted-71827]

Hi!
Great to know that the link helped! As Walker mentioned before,

While the how-to on the ScienceBuddies site calls for the use of 9 volt batteries to power the elecrophoresis, you can get faster results with a simple DC power supply you can build yourself. An example of how to build a power supply for electrophoresis is at

http://www.science-projects.com/PowerSupply.htm

You could use this apparatus to separate DNA out in agarose gels and visualize the DNA using a chemical stain. (You'd need to purchase agarose and the DNA stain) You could see by the strength of the staining and the relative size of the DNA pieces in the gel how much DNA degradation had occurred in your samples.

I think that one option for gathering your data would be in a table, which you can eventually turn into a graph if you would like. Things that you might want to include in the table is the relative size of the DNA pieces as they flow through the gel (you must come up with some type of standardized measurement for this) and/or the thickness of the band. One thing to be careful about is that relative thickness is easy to see when you have multiple bands, but it may be harder to compare the thickness of the bands across different samples one after the other. Hope this helps!

[charlescullen28]

yes the information helped a lot again, but i think i have to add another bit to this...... i'm in an international school in china so....... buying these things could be hard.... well at least the things on the link that you sent but it still helped so thanks again

[zoya12345ttt]

please suggest me how should i do my graph for this topic pls
i searched everywhere i dint get any graph
so pls say me how should i do the graph

[zoya12345ttt]

plsss say me how should to do grapph for this topic pls pls

[SciB]

Your question is confusing. Did you do this project? If so, please explain what you did and answer on a NEW thread. Do not put your questions on someone else's posting. And please give us more details describing exactly what problem you are having.

Sybee

[zoya12345ttt]

Oh im sorry.
OK my project is can DNA be separated from other cell components and survive.
so i need a graph related to this topic.
please suggest me some graphs related to this topic.

[SciB]

Can you please explain exactly what you DID. We cannot help you if you do not take the time to tell us about your specific project and not just the title.

If you read the previous posts in this thread the expert explained one way to measure how well DNA survives--by measuring the size of the DNA strands using gel electrophoresis. He provided many links and Scibuddies guides to how to do this. Did you read all that? Do you understand it? If not then ask questions and we will answer them for you.

Since I don't know exactly how you isolated the DNA or from what you isolated it and how you treated it afterwards, I can only tell you that the 'survival' measurement, whatever it is, will go on the y-axis of your graph and the x-axis will be time, probably in days.

If you could please describe your experiment in detail and tell how you measured DNA 'survival' we can help you.

Sybee