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How do I isolate DNA?

J

JCE10

Member
So I'm looking to extract some DNA and in the future, when I make more than minimum wage, I would like to get it sent off and get my DNA sequenced (the right word?). I was wondering what was the best way to conserve my DNA outside of my body. I heard freezing it would break down the cells and destroy or currupt the DNA. How about a piece of hair? Does that offer enought o do a squence? I see most places just swab your mouth but if I kept that, it would get kind of disgusting and I think the bacteria would eat away at whats left.

I need answers people! =)

Thank you all!
 
DNA is destroyed by (A) time and (B) temperature. The longer you want it to last, the colder it must be. I suggest a -70°C freezer. I think you can find one for just under $2000.

Freezing/thawing cells may cause the cells to rupture (especially after repeated freeze/thaw cycles), but it isn't too harsh on the DNA.

The human DNA is about 3 billion DNA bases long. If you shop around, you can get DNA sequenced for ~5 cents per base. So we are talking $150 million dollars. Maybe you can get a quantity discount.

As for the DNA source, I think you can find some cells just about anywhere on your body. Pick something off and you are done. Note: not everything you pick off will have DNA, so maybe take a few samples from all over.

 
assuming you're still alive when you collect said money, I imagine it wouldn't be too difficult to get such a sample of DNA when you need it. Making this whole isolating/storing question a moot point.
(it wouldn't even be difficult if you were dead 😛)
 
How are scientists able to get such good (relatively speaking) samples of DNA from really ancient bones?

And can DNA be found in hair?
 
How to Extract DNA from Anything Living
You mean I can see it? How?
Just follow these 3 easy steps:
Detergent
eNzymes (meat tenderizer)
Alcohol
It's that simple? Tell me more!

First, you need to find something that contains DNA. Since DNA is the blueprint for life, everything living contains DNA.

For this experiment, we like to use green split peas.
EDIT: (For the challenged of ATOT: You would of course substitute inner cheek scrapings in place of the peas)
But there are lots of other DNA sources too, such as:
? Spinach
? Chicken liver
? Onions
? Broccoli
Here's the fun part. Put in a blender:
? Your DNA source (about 100ml or 1/2 cup of split peas)
? A large pinch of table salt (less than 1ml or 1/8 teaspoon)
? Twice as much cold water as the DNA source (about 200ml or 1 cup)

Blend on high for 15 seconds.
The blender separates the pea cells from each other, so you now have a really thin pea-cell soup. Because this step is pretty messy, certain sources of DNA should not be used, such as:
? Your family pet, Fido the dog
? Your little sister's big toe
? Bugs you caught in the yard
And now, those 3 easy steps:

1. Pour your thin pea-cell soup through a strainer into another container (like a measuring cup).
How much pea soup do you have? Add about 1/6 of that amount of liquid Detergent (about 30ml or 2 tablespoons) and swirl to mix. Let the mixture sit for 5-10 minutes.
Pour the mixture into test tubes or other small glass containers, each about 1/3 full.

Try one of these detergents or whatever you have on hand.

Why am I adding detergent?
2. Add a pinch of enzymes to each test tube and stir gently. Be careful! If you stir too hard, you'll break up the DNA, making it harder to see.
Use meat tenderizer for enzymes. If you can't find tenderizer, try using pineapple juice or contact lens cleaning solution.

What is an enzyme?
3. Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same amount of alcohol in the tube as pea mixture.

DNA will rise into the alcohol layer from the pea layer. You can use a wooden stick or other hook to draw the DNA into the alcohol.
What is that stringy stuff?

Alcohol is less dense than water, so it floats on top. Since two separate layers are formed, all of the grease and the protein that we broke up in the first two steps and the DNA have to decide:

"Hmmm...which layer should I go to?"
This is sort of like looking around the room for the most comfortable seat. Some will choose the couch, others might choose the rocking chair.

In this case, the protein and grease parts find the bottom, watery layer the most comfortable place, while the DNA prefers the top, alcohol layer.
DNA is a long, stringy molecule that likes to clump together.

Congratulations! You have just completed a DNA extraction!
Now that you've successfully extracted DNA from one source, you're ready to experiment further. Try these ideas or some of your own:
? Experiment with other DNA sources. Which source gives you the most DNA? How can you compare them?
? Experiment with different soaps and detergents. Do powdered soaps work as well as liquid detergents? How about shampoo or body scrub?
? Experiment with leaving out or changing steps. We've told you that you need each step, but is this true? Find out for yourself. Try leaving out a step or changing how much of each ingredient you use.
? Do only living organisms contain DNA? Try extracting DNA from things that you think might not have DNA. Frequently Asked Questions
1. I'm pretty sure I'm not seeing DNA. What did I do wrong?
First, check one more time for DNA. Look very closely at the alcohol layer for tiny bubbles. Often, clumps of DNA are loosely attached to the bubbles.
If you are sure you don't see DNA, then the next step is to make sure that you started with enough DNA in the first place. Many food sources of DNA, such as grapes, also contain a lot of water. If the blended cell soup is too watery, there won't be enough DNA to see. To fix this, go back to the first step and add less water. The cell soup should be opaque, meaning that you can't see through it.
Another possible reason for not seeing any DNA is not allowing enough time for each step to complete. Make sure to stir in the detergent for at least five minutes. If the cell and nuclear membranes are still intact, the DNA will be stuck in the bottom layer. Often, if you let the test tube of pea mixture and alcohol sit for 30-60 minutes, DNA will precipitate into the alcohol layer.
2. Why does the DNA clump together?
Single molecules of DNA are long and stringy. Each cell of your body contains six feet of DNA, but it's only one-millionth of an inch wide. To fit all of this DNA into your cells, it needs to be packed efficiently. To solve this problem, DNA twists tightly and clumps together inside cells. Even when you extract DNA from cells, it still clumps together, though not as much as it would inside the cell.
Imagine this: the human body contains about 100 trillion cells, each of which contains six feet of DNA. If you do the math, you'll find that our bodies contain more than a billion miles of DNA!
3. Can I use this DNA as a sample for gel electrophoresis?
Yes, but all you will see is a smear. The DNA you have extracted is genomic, meaning that you have the entire collection of DNA from each cell. Unless you cut the DNA with restriction enzymes, it is too long and stringy to move through the pores of the gel; instead, all you will end up seeing is a smear.
4. Isn't the white, stringy stuff actually a mix of DNA and RNA?
That's exactly right! The procedure for DNA extraction is really a procedure for nucleic acid extraction. However, much of the RNA is cut by ribonucleases (enzymes that cut RNA) that are released when the cells are broken open.




 
Originally posted by: JCE10
How are scientists able to get such good (relatively speaking) samples of DNA from really ancient bones?

And can DNA be found in hair?
Click to expand...

not in hair, but if the root is intact, you can find cells there ... look

in ancient bones, first they have to go digging for tissue, but even if they find anything, I imagine the full DNA is NEVER intact. Maybe a few genes here or there. Or maybe some hundred-or-so, base pair sequences that are useful for comparison to modern species.
Do you have any reference links to your ancient DNA quesiton?
 
Originally posted by: JCE10
How are scientists able to get such good (relatively speaking) samples of DNA from really ancient bones?

And can DNA be found in hair?
Click to expand...

I think DNA can indeed be found in hair but IANAB.

Can they really get good DNA from ancient bones? This isn't jurassic park, I don't think they have and good samples from dinosaurs...

Does bone even have DNA? Marrow does, but fossilized bone don't contain the marrow.

/edit: TheChort you answered almost all my questions! Does bone contain DNA, or only marrow?
 
Originally posted by: JCE10
So I'm looking to extract some DNA and in the future, when I make more than minimum wage, I would like to get it sent off and get my DNA sequenced (the right word?). I was wondering what was the best way to conserve my DNA outside of my body. I heard freezing it would break down the cells and destroy or currupt the DNA. How about a piece of hair? Does that offer enought o do a squence? I see most places just swab your mouth but if I kept that, it would get kind of disgusting and I think the bacteria would eat away at whats left.

I need answers people! =)

Thank you all!
Click to expand...

You do realize that youll have the same dna later, right?
 
DNA is pretty stable, so any sample that contains some cells can just be chucked into a freezer. Blood, a hunk of skin, etc. A sperm sample might be the best way, honestly - large number of cells, high percentage of DNA by weight.

OTOH, I don't know why you feel a need to get the DNA now and sequence it later. I also don't know what information you hope to get from sequencing it, either. I was going to sequence some of my own DNA at one point, but the practical need went away, and with it, the motivation.
 
Originally posted by: dullard
DNA is destroyed by (A) time and (B) temperature. The longer you want it to last, the colder it must be. I suggest a -70°C freezer. I think you can find one for just under $2000.

Freezing/thawing cells may cause the cells to rupture (especially after repeated freeze/thaw cycles), but it isn't too harsh on the DNA.

The human DNA is about 3 billion DNA bases long. If you shop around, you can get DNA sequenced for ~5 cents per base. So we are talking $150 million dollars. Maybe you can get a quantity discount.

As for the DNA source, I think you can find some cells just about anywhere on your body. Pick something off and you are done. Note: not everything you pick off will have DNA, so maybe take a few samples from all over.
Click to expand...


In the labs I have worked in, and common experience all over seems to dictate, that a simple 4 degree (Celsius) fridge is sufficient for storing DNA for long periods of time. Hell, we'll leave tubes of purified DNA out at room temperature for a few days and it's fine.

The problem with freezing it is not the act of freezing, but constantly thawing and refreezing. In purified samples, this will nick the DNA (it is also important to consider which buffer this is stored in--DI water is perfectly fine, actually)--but that's for purified DNA

If you want a "messy" sample:

--rinse your mouth with some really, really salty water a few times, and spit contents into a cup (you want a conical tube with screw cap preferrably, 50mL is perfect)
You're collecting cheek cells here. the salt is simply to loosen them.

--add common detergent (I can't give you ratio to sample...as this won't be a very good extraction anyway) shake vigorously
detergent breaks the cell membranes of your cheek cells, and releases the contents out into solution

--now add 100% isoproponol. regular rubbing alcohol works fine
This isolates the dna from all of the other junk (more or less) you will notice a milky precipitate form in the solution. this is Your DNA.


--you would have to wash it with 70% ethanol after this (not sure how to separate if you don't have a centrifuge). but, if you were able to wash a few times, and separate the precipitate from solution, then you would just pour off the liquicd, and let the white "pellet" air dry. once the ethanol has completely evaporated, you could resuspend with DI water.


common misconception from shows like CSI is that an eyelash, or any hair will provide a DNA sample. For that to work, and to be usable, the follicle has to be attached to the hair sample. This only occurs if the hair has been pulled out. One that simply falls out naturally will be almost impossible to isolate DNA from.


Another, much more useful solution for you: Northwestern's NuGene program (of which I am loosely associated through department), is compiling a genomic database to be used as a resource to study and identify genetic diseases. They obtain their samples from any willing patient that contributes a blood sample during any scheduled Hospital visit at Northwestern. Names are completely anonymous, as the intent is to create a searchable database for research labs all over the world. I believe they are ~3,000 individuals and 3 years into their 10,000 individual/10 year plan. I'm not sure about the access to personal data that is granted to participants, but it is my belief that this is/or will soon be possible....

gl with home experimentation...and please do provide pics of your home dna isolation 🙂
 
Originally posted by: Atheus
Originally posted by: JCE10
How are scientists able to get such good (relatively speaking) samples of DNA from really ancient bones?

And can DNA be found in hair?
Click to expand...

I think DNA can indeed be found in hair but IANAB.

Can they really get good DNA from ancient bones? This isn't jurassic park, I don't think they have and good samples from dinosaurs...

Does bone even have DNA? Marrow does, but fossilized bone don't contain the marrow.

/edit: TheChort you answered almost all my questions! Does bone contain DNA, or only marrow?
Click to expand...

Bone itself doesn't have DNA, you're right, it's only the marrow
But I'm thinking the same thing you are ... I can't think of any cases where fossilized bones have given us any workable amounts of DNA. That's why I was asking the OP for a source
 
Originally posted by: TheChort
Originally posted by: Atheus
Originally posted by: JCE10
How are scientists able to get such good (relatively speaking) samples of DNA from really ancient bones?

And can DNA be found in hair?
Click to expand...

I think DNA can indeed be found in hair but IANAB.

Can they really get good DNA from ancient bones? This isn't jurassic park, I don't think they have and good samples from dinosaurs...

Does bone even have DNA? Marrow does, but fossilized bone don't contain the marrow.

/edit: TheChort you answered almost all my questions! Does bone contain DNA, or only marrow?
Click to expand...

Bone itself doesn't have DNA, you're right, it's only the marrow
But I'm thinking the same thing you are ... I can't think of any cases where fossilized bones have given us any workable amounts of DNA. That's why I was asking the OP for a source
Click to expand...

You can get DNA from bone, exclusive of the marrow. Not easily, but it's been done.
 
Originally posted by: zinfamous
In the labs I have worked in, and common experience all over seems to dictate, that a simple 4 degree (Celsius) fridge is sufficient for storing DNA for long periods of time. Hell, we'll leave tubs of purified DNA out at room temperature for a few days and it's fine.
Click to expand...
Yes, it is fine at 4°C. I regularly ship out DNA without any form of cooling and it arrives just fine halfway around the world. I thought the sarcasm was evident in my post. I did delete the wink though in one edit. With a $150 million sequencing cost, the cost of storage should be easy to swing.
 
Originally posted by: dullard
Originally posted by: zinfamous
In the labs I have worked in, and common experience all over seems to dictate, that a simple 4 degree (Celsius) fridge is sufficient for storing DNA for long periods of time. Hell, we'll leave tubs of purified DNA out at room temperature for a few days and it's fine.
Click to expand...
Yes, it is fine at 4°C. I regularly ship out DNA without any form of cooling and it arrives just fine halfway around the world. I thought the sarcasm was evident in my post. I did delete the wink though in one edit. With a $150 million sequencing cost, the cost of storage should be easy to swing.
Click to expand...


ya...some PIs will ship DNA in a small envelope (Fedex). All you need to do is take a kimwipe (tissue paper), and blot it with a purified DNA solution, and let the sample dry. Could look like a CIA microdot...Just dissolve teh paper in water and...viola!

Is the DNA that you ship without ice pacs in precipitate form?
 
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