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DNA Preparation
Do you want to study DNA?
You are a researcher, a zoologist, a medical doctor, or a policeman.
You have a sample of
plant, blood, fly, sperm, biopsy, a culture of bacteria or an onion.
The only thing you want from all that is
the pure DNA...
Well, you have work to do.
Table of Contents
- What's in a sample?
- Get rid of that junk! DNA purification.
- Protect your DNA.
- Now what! Why do people want DNA?
What's in a sample!
When we studied "The Cell", we learned
that DNA is enclosed in a nucleus, which is itself in a cell. The DNA of
an individual is not readily available, and in order to study it, there
is a considerable number of things which need to be done.
Your sample can be a whole (small) animal, like an aphid, or a fly, if
you work in agriculture. A lot of the tissues in the housefly are hard,
and waterproof. They need to be broken physically before the purification
starts. During purification, proteins, carbohydrates and lipids will be removed from the sample to leave only DNA.
For some DNA projects, like the study of a special gene, you might want
to prepare RNA
first. When you have your messenger
RNA, it is easy to make what we call cDNA, which is DNA made from mRNA.
cDNA is used a lot in research, to study genes, and proteins.
DNA Purification
In research, in medical labs, or in police forensic labs, the sample is often very small, and some of the steps in the purification of DNA involve the use of dangerous components.
In a classroom, your sample, usually some cress, or onion, has a lot of DNA, and is easy to extract.
| Step | What to do | Why? |
| 1 | Grind the material. Use a pestle
and mortar. It is best to work on ice. |
It is important to start breaking down the structures as much as possible. When you break down cellular structures, proteins could come into contact with DNA and degrade it. That is why it is important to keep your sample cold. |
| 2 | Incubate at 60°C, with a solution of detergents and salt. | The detergent will destroy all the lipid membranes while the sodium choride causes DNA to coagulate. The high temperature prevents proteins from degrading DNA. |
| 3 | The mixture probably looks disgusting now. Put your tube on ice. | The long jelly like mucus is actually what DNA looks like when in solution at high concentration. It makes long threads. Cooling down the sample helps prevent the DNA from breaking down. |
| 4 | The mixture is blended and filtered. The filtrate contains proteins and DNA. | The filtration helps remove all the big pieces that the preceding steps have not. |
| 5 | Add some protease to the solution. |
The protease will degrade histone proteins
and the enzymes that could degrade DNA. |
| 6 | Carefully layer on some chilled ethanol. | DNA will precipitate. You can see the white precipitate forming at the interface. You can actually collect the DNA with a glass rod. |
| 7 | You can rinse your DNA in chilled ethanol, and dry it on the bench. Dilute the DNA pellet in distilled sterile water. | The DNA is best kept in a freezer. In a
research lab, we add some EDTA at a very low concentration. EDTA protects
DNA from attacks by enzymes specific to DNA. |
This method is very simple and very easy: it can be done in a school
laboratory. It does not use any dangerous solutions, so one could actually
prepare DNA in a kitchen. I have described another
example on another page... When DNA needs to be extracted from
small samples, or from tiny animals, then it is important not to lose any.
The methods employed are more complex. Scientists use a phenol
/ chloroform solution to destroy proteins and obtain a very clean solution
of DNA. In the laboratory we also use a centrifuge
which spins at high speeds and DNA is pelleted at the bottom of the
tube. Centrifugating is a quick and efficient way to recover all, or most of the DNA from a suspension.
Biotechnology companies provide kits to simplify DNA extraction even more. They usually contain beads that bind to DNA very specifically. The beads are collected, alond with bound DNA either by centrifugation, or with a magnet, depending on the kit.
Keep your DNA safe!
DNA is a long molecule, and is prone to breaking down easily. It does not break down spontaniously, but there are a lot of enzymes around which are specialized in breaking down DNA. Your hands are probably covered with them, and although you may not realize it.
- It is important to use gloves at all times when working with DNA.
- It is important to use sterile material especially the material used for the last steps. Use sterile pipettes and tubes.
- Work on ice. Keep all your solutions cold (exept the detergent solution, as it precipitates in the cold).
- Store your DNA in TE (a buffer (Tris) containing also some EDTA), and in the freezer.
- If you do not have a freezer, use your DNA quickly!
And now, let's do something with our DNA!
Well, we now have a nice, neat little tube with a DNA solution in it. What can we do with it?
- Lets measure its concentration:
- Can we see what we have?
- Cut Cut Cut
- PCR , sequencing and other methods.
DNA has the property of changing the optical density of water. In plain English, it colours water, but in a colour we can't see. However, optical density can be measured with a special machine, a spectrophotometer. As there is a simple relation between optical density and the concentration of DNA, it is very easy to measure the concentration of DNA
In order to see what size fragments we have obtained, we have to make a gel, pour our sample DNA into it, and stain it. This is explained further in the next chapter.
We use special tools (enzymes) to work with DNA. Some, as we will soon see, help us to gather a lot of information about DNA. We can cut DNA where we want, and re-assemble it as well.
In order to get all the information we want, perform cloning, or find out all the information contained in DNA, more and more techniques are used. Some are explained in these chapters...
DNA Preparation on Wikipedia
