How to see DNA
August 29th, 2009
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Author: Heredity, genes, DNA ... It seems that these words have long ceased to be scientific terms, entered into everyday life and is now familiar to every senior, not to mention the students. But there was no DNA, most of us have never seen, though to see it - it is quite real, even in the home. In one of the genetic laboratory on the wall, for example, that the instruction:
- Find something that contains a lot of DNA. For example, green peas (but may be chicken liver, herring roe, or onion).
- Put in a blender about 100 ml (half a cup) of this product, add 1 / 8 teaspoon of salt and 200 ml (cup) of cold water. Whips within 15 seconds. Mixer «cook» you pea-cell soup.
- Strain
the mixture through a strainer or a piece of nylon (stockings quite fit). In the obtained pulp, add 1 / 6 of its number (it will be about 2 tablespoons) of liquid detergent (for dishes, for example) and stir well. Leave on for 5-10 minutes.
Pour the liquid in test tube or other glass vessel, so that each was filled with no more than a third volume.
Add to each tube for a bit, or the juice squeezed out of a pineapple, a contact lens solution and gently shake it, turning and bending the tube (if you shake too vigorously, break up DNA and did not see it).
Tilt the tube and slowly pour into it a little bit of ethyl alcohol, that it formed a layer on top of pea mixture. Leite, while a mixture of alcohol and not be a tie. DNA pops up in the form of flakes.
Wooden stick (pencil) to catch them and examine under a microscope.
Of course, for researchers this instruction - to some extent a joke and none of them in this way the DNA does not evolve, but in the meantime if you really use it, then everything will turn out! Output DNA is, however, is small, and the substance - not particularly clean, but seen through a microscope long thin filaments - crystals of DNA technology - it is quite possible.
What happens with green peas or chicken liver in the process described manipulation and why eventually the DNA is separated from all other substances, which in a great many cells?
1. Select object
DNA is known to have in each cell, and thus make it possible from any fabric - even from animal bones, fish scales, or wood, where the cells are not so much in comparison with the volume of extracellular substances.
In all tissues of both animals and plants, DNA is usually the same. Differences between the tissues that in some of them more than the substance of heredity almost nothing (herring roe), while in others, such as bone tissue, DNA content is relatively low. In addition, there are tissues in which cells have a doubled set of chromosomes (tetraploid to include, in particular, liver cells), and therefore the DNA in them two times more than all the rest. In seed plants relative, DNA content higher than in the stem, and from the young growing shoots it is possible to allocate substantially more than the same volume of a piece of lignified stem.
In general, if the researcher is not worth a special problem, it tries to select for tissue in which little intercellular substance and a lot of cells themselves. It is desirable that the fabric easily fall into these components, and the cells were not overloaded with protein (like muscle), lipids (as lipid) or polysaccharides (such as brain cells).
2. Fractions tissue cells
In blender fabric from which we are going to get the substance of heredity, is divided into separate cells: in order to mechanically break the connection between them is usually required much less effort than to damage the cell itself. And as with our method of DNA isolation require more or less whole, intact cells, it is clear that canned peas or salted herring for such experiments do not work - better to take something frozen, if you are sure that the product is thawed during storage several times.
A little salt should be added to the solution so that the cells do not burst ahead of time: the internal pressure of the contents of the cell membrane to equalize the pressure inside a saline solution from the outside.
3. Macromolecule releases
As for filtration, it is necessary to mechanically remove from the cell suspension of various impurities, including large pieces of fabric - all the same substances that we are going to handle the mixture will not be able to penetrate deep into these conglomerates, and to extract DNA will be useless.
A process must be received by the cells in the first place, some detergent. Tool "Ferry", capable, according to advertising, it is easy to clean the most greasy dishes, fit and in order to make many holes in the lipid membrane of both the cell and its nucleus. If no liquid detergent, you can make a concentrated solution of detergent - is also nice.
As a result of processing all the cellular contents and would be hung out in solution, which is done with this very sticky, viscid and significantly more transparent than was the cell suspension. Changing the consistency of the solution - a sure sign that the lysis was successful.
4. Everything is clear and no comments --
do not pour too much mud in a container, there is still much to pour, in addition, if the mixture will be in excess, it will be difficult to mix.
5. Freed from proteins
Why just not in our mix! However, the proteins here - most of all, and they form the most stable complexes with DNA. There are techniques where proteins are removed from the solution in several stages. For example, some of them easily denatures and precipitates by adding concentrated solutions of salts. Under laboratory conditions, these methods work fine, but researchers are exempt from the draft, putting the tube for several minutes in a centrifuge. After that, all more or less large cell debris, denatured proteins and other impurities are at the bottom, forming a very dense cake, and poured into another test tube supernatant containing mainly nucleic acids - DNA and RNA - is not working. But at home, this purification step we have to miss, we are interested in donating part of the substance - it will remain a "prisoner of the protein.
We will immediately proceed to the purification of DNA from residual proteins with special enzymes that can destroy these molecules. It is such a substance contains pineapple juice. They themselves - the same protein, so the pineapple, from which the pressed juice must be fresh: the enzyme is not the slightest chance to survive intact in compote or canned food. As a solution for cleaning lenses, if you intend to use it - do not forget to put a pill to remove protein deposits! Themselves, the solutions for storing contact lenses are no active ingredients do not contain - otherwise our eyes and not be faint.
The fact that the enzymes worked, you can try to reduce the viscosity of the solution. If not, place the mixture in a warm place (about 37 ° C) for half an hour, sometimes you may need to add more pineapple juice or a solution for cleaning lenses.
6. DNA precipitated from solution
Now DNA floating in the solution by itself. Proteins do not cling to it, though the wreckage of various molecules in the mixture is still a lot. Under laboratory conditions, these unwanted parts removed, thoroughly mixing the solution with phenol and / or chloroform. Organic solvents that can pick up proteins on a "heavier than water, and therefore the subsequent stratification of the mixture in a centrifuge, they sink to the bottom. After centrifugation the bottom of the tube are phenol and / or chloroform with dissolved proteins in them, and at the top - the water phase containing DNA. The aqueous phase is collected in a separate tube and more are already working with a relatively clean solution.
In the absence of the centrifuge and organic solvents, which requires work to the same special security measures, this stage of treatment at home have missed and to precipitate the DNA directly from the "dirty" solution.
We note at once - to replace the ethyl alcohol vodka or spirits can not be: if the concentration of alcohol is low and falls when mixed with an aqueous phase up to 60-65% of DNA in the crystalline state can not pass. Partly for this reason to pour alcohol into a test tube with the DNA-containing mixture should be carefully layering it on top. Then the lower layers of partially mixed with alcohol solution of DNA, will begin the process of crystallization of nucleic acids, and they come to the surface (where alcohol is more concentrated) in the form of flakes.
If you pour alcohol on top do not get all hopelessly mixed up, then the small amount of ethanol you do not succeed, but when a large starts to crystallize not only DNA: a precipitate fall out and the remnants of proteins, and something else from the original content of the cells.
7. What have we got?
Pure crystals of DNA are like balls of tangled fibers, but we must not forget that you can see the crystals of substance rather than its macromolecules, and say to their appearance, which contains genes isolated nucleic acid by you, of course, impossible. To find out, would have to dissolve the DNA. However, "read" the sequence of nucleotides in the home, alas, is impossible: it requires not only special devices, but also expensive reagents.
However, if you are already well-reviewed crystals and they managed to dry out, you can observe how the DNA is dissolved. It is the beginning swells and becomes jelly-like jellyfish, and only a few days later, the solution is homogeneous. The process can be accelerated if the tube often shake.
1. Select object
DNA is known to have in each cell, and thus make it possible from any fabric - even from animal bones, fish scales, or wood, where the cells are not so much in comparison with the volume of extracellular substances.
In all tissues of both animals and plants, DNA is usually the same. Differences between the tissues that in some of them more than the substance of heredity almost nothing (herring roe), while in others, such as bone tissue, DNA content is relatively low. In addition, there are tissues in which cells have a doubled set of chromosomes (tetraploid to include, in particular, liver cells), and therefore the DNA in them two times more than all the rest. In seed plants relative, DNA content higher than in the stem, and from the young growing shoots it is possible to allocate substantially more than the same volume of a piece of lignified stem.
In general, if the researcher is not worth a special problem, it tries to select for tissue in which little intercellular substance and a lot of cells themselves. It is desirable that the fabric easily fall into these components, and the cells were not overloaded with protein (like muscle), lipids (as lipid) or polysaccharides (such as brain cells).
2. Fractions tissue cells
In blender fabric from which we are going to get the substance of heredity, is divided into separate cells: in order to mechanically break the connection between them is usually required much less effort than to damage the cell itself. And as with our method of DNA isolation require more or less whole, intact cells, it is clear that canned peas or salted herring for such experiments do not work - better to take something frozen, if you are sure that the product is thawed during storage several times.
A little salt should be added to the solution so that the cells do not burst ahead of time: the internal pressure of the contents of the cell membrane to equalize the pressure inside a saline solution from the outside.
3. Macromolecule releases
As for filtration, it is necessary to mechanically remove from the cell suspension of various impurities, including large pieces of fabric - all the same substances that we are going to handle the mixture will not be able to penetrate deep into these conglomerates, and to extract DNA will be useless.
A process must be received by the cells in the first place, some detergent. Tool "Ferry", capable, according to advertising, it is easy to clean the most greasy dishes, fit and in order to make many holes in the lipid membrane of both the cell and its nucleus. If no liquid detergent, you can make a concentrated solution of detergent - is also nice.
As a result of processing all the cellular contents and would be hung out in solution, which is done with this very sticky, viscid and significantly more transparent than was the cell suspension. Changing the consistency of the solution - a sure sign that the lysis was successful.
4. Everything is clear and no comments --
do not pour too much mud in a container, there is still much to pour, in addition, if the mixture will be in excess, it will be difficult to mix.
5. Freed from proteins
Why just not in our mix! However, the proteins here - most of all, and they form the most stable complexes with DNA. There are techniques where proteins are removed from the solution in several stages. For example, some of them easily denatures and precipitates by adding concentrated solutions of salts. Under laboratory conditions, these methods work fine, but researchers are exempt from the draft, putting the tube for several minutes in a centrifuge. After that, all more or less large cell debris, denatured proteins and other impurities are at the bottom, forming a very dense cake, and poured into another test tube supernatant containing mainly nucleic acids - DNA and RNA - is not working. But at home, this purification step we have to miss, we are interested in donating part of the substance - it will remain a "prisoner of the protein.
We will immediately proceed to the purification of DNA from residual proteins with special enzymes that can destroy these molecules. It is such a substance contains pineapple juice. They themselves - the same protein, so the pineapple, from which the pressed juice must be fresh: the enzyme is not the slightest chance to survive intact in compote or canned food. As a solution for cleaning lenses, if you intend to use it - do not forget to put a pill to remove protein deposits! Themselves, the solutions for storing contact lenses are no active ingredients do not contain - otherwise our eyes and not be faint.
The fact that the enzymes worked, you can try to reduce the viscosity of the solution. If not, place the mixture in a warm place (about 37 ° C) for half an hour, sometimes you may need to add more pineapple juice or a solution for cleaning lenses.
6. DNA precipitated from solution
Now DNA floating in the solution by itself. Proteins do not cling to it, though the wreckage of various molecules in the mixture is still a lot. Under laboratory conditions, these unwanted parts removed, thoroughly mixing the solution with phenol and / or chloroform. Organic solvents that can pick up proteins on a "heavier than water, and therefore the subsequent stratification of the mixture in a centrifuge, they sink to the bottom. After centrifugation the bottom of the tube are phenol and / or chloroform with dissolved proteins in them, and at the top - the water phase containing DNA. The aqueous phase is collected in a separate tube and more are already working with a relatively clean solution.
In the absence of the centrifuge and organic solvents, which requires work to the same special security measures, this stage of treatment at home have missed and to precipitate the DNA directly from the "dirty" solution.
We note at once - to replace the ethyl alcohol vodka or spirits can not be: if the concentration of alcohol is low and falls when mixed with an aqueous phase up to 60-65% of DNA in the crystalline state can not pass. Partly for this reason to pour alcohol into a test tube with the DNA-containing mixture should be carefully layering it on top. Then the lower layers of partially mixed with alcohol solution of DNA, will begin the process of crystallization of nucleic acids, and they come to the surface (where alcohol is more concentrated) in the form of flakes.
If you pour alcohol on top do not get all hopelessly mixed up, then the small amount of ethanol you do not succeed, but when a large starts to crystallize not only DNA: a precipitate fall out and the remnants of proteins, and something else from the original content of the cells.
7. What have we got?
Pure crystals of DNA are like balls of tangled fibers, but we must not forget that you can see the crystals of substance rather than its macromolecules, and say to their appearance, which contains genes isolated nucleic acid by you, of course, impossible. To find out, would have to dissolve the DNA. However, "read" the sequence of nucleotides in the home, alas, is impossible: it requires not only special devices, but also expensive reagents.
However, if you are already well-reviewed crystals and they managed to dry out, you can observe how the DNA is dissolved. It is the beginning swells and becomes jelly-like jellyfish, and only a few days later, the solution is homogeneous. The process can be accelerated if the tube often shake.
Posted in DNA