MITOCHONDRIAL DNA ISOLATION

PROCEDURE

Grind in mortar and pestle or Waring blender with 5-7 volumes buffer A per g tissue. Use MCE at 350 l/L, and if necessary, with 5 ml 1 M DIECA/L.
Squeeze through cheesecloth, two layers of Miracloth.
Centrifuge 10 min at 1000 g
Decant supernatant and centrifuge 10 min at 15,900 g.
Resuspend each pellet in a few drops of buffer G with paint brush; combine; bring to about 10 ml/50 g, 15 ml/75 g.
Centrifuge 10 min at 1000 g; pour off most; swirl pellet to remove fluffy layer; combine.
Bring supernatant to 10 mM MgCl2 (100 l 1M/10 ml). Bring to 20 g DNase/ml (100 l 2mg/ml/10 ml).
60 min. 4 C.
Underlay shelf buffer, 20 ml/10-15 ml; always use 20 ml or more.
Centrifuge 20 min at 12000 g.
Resuspend in small volume shelf buffer with brush; bring to about 10 ml/50-100 g.
Centrifuge 10 min at 15900 g.
Resuspend pellets in NN (lysis) buffer (4-5 ml/50-75 g).
Add SDS to 0.5% (250 l of 10%/5 ml NN). Swirl thoroughly.
Add proteinase K to 100 g/ml (25 l of 20 mg/ml/5 ml NN). Swirl gently.
60 min. 37 C.
Add equal volume of 3:1 water-saturated phenol, chloroform-isoamyl alcohol mixture. Emulsify ca. 5 min.
Centrufuge 10 min at 7000 g.
Collect supernatant; repeat 17 and 18: 3 total extractions.
Final supernatant; add 0.1 volume 8 M Ammonium acetate; then add 2 volumes of absolute ethanol.
60 min, -80 C; 10 min at 8000-9000 g; drain; add equal volume 70% ethanol; let sit 10 min; 10 min at 8000-9000 g; drain dry. Vacuum dry pellet, 30 min. Two small corex tubes are better than one 30 ml Corex.
Add 100-500 l 0.1X NTE, 10 l RNase mixture. Typically use 500 l per 50 g tissue.
Hydrate 30 min., 37 C.

Column Chromatography

Column In TLC, the stationary phase is a thin layer of silica gel or alumina in glass, metal or plastic plate. Column chromatography works in a much larger scale for the packaging of the same material in a vertical column of glass. Different sizes of chromatography columns used, and if you have the link at the bottom of the section Organic Chemistry site, at the University of Colorado, you will find images of several columns. In the school laboratory, it is often convenient to use as an ordinary burette column chromatography.
Using column Suppose you want to separate a mixture of two compounds of colors - yellow, blue. The mixture looks green. You want to make a concentrated solution of the mixture, preferably on the solvent used in Stoubtsy. The first time you turn on the tap in order to have a solvent in the column to drain so that at the same level with the top of the packing material, then carefully add the solution to the top of the column. Then open the valve again to the color mixing is absorbed in the upper packaging material, so it may look as follows:
Explaining what happens This means that you have read the explanation of what happens during the thin-layer chromatography. If you do not, go to the first in the top of the page and return to this issue later.
Blue, obviously, more polar than the yellow - it even has the possibility of hydrogen bonding.You can say this because the blue does not travel through the column very quickly. This means that it must absorb more energy for silica or alumina in yellow. Less polar yellow to spend most of their time in the solvents and, thus, is washed through the column much faster.
Washing process connection through the column using a solvent known as elution. Thinner sometimes referred to as eluent.
What to do if you want to pick up the blue compound, too?
Will take years to wash the blue through rate that moves at the moment! However, there is no reason why you can not change the course of elution solvent.
Suppose that we replace the solvent was used for the polar solvent, the yellow once again were collected. This will have two consequences, both of which accelerate the blue through the column.
Polar solvents compete for a place in the silica gel or aluminum with blue. Any space is temporarily occupied by solvent molecules on the surface of the stationary phase is not available for blue molecules stick, and it is usually going to hold them together with solvent.
There will be more involvement between the polar solvent molecules and polar molecules in blue.It will seek to attract any blue molecules in the stationary phase of the decision once again.
The end result is that the more polar solvents, the blue to spend more time on the decision and, thus, is moving faster.
Why not use this alternative solvent in the first place? The answer is that when both compounds in a mixture of rapid passage through the column from the beginning, would probably not be as good a separation.
What to do if all they have a mixture of colorless? If you use column chromatography for purification of organic food preparation, it is likely that the product you want to be colorless, colored, even if one or more impurities. Assume that the worst of all, colorless.
As you know, when the creature that he had reached the bottom of the column?
There are no quick and easy way to do it! What it does is collect what comes out of the bottom of the column in a series of labeled tubes. What is the size of each sample, obviously, depend on how popular column - you can collect samples of 1 cm3 or 5 cm3 samples or all that is necessary.
Can then take one drop of each solution and make a thin layer chromatogram it. You can put a drop on the base with a drop of pure sample of a substance does. By doing this several times, you can determine which of the samples collected in the bottom of the column containing the desired product, and only the desired product.
Once you know it, you can combine all the samples containing pure product, and then remove the solvent. (How not to be separated from the solvent product is not directly related to this topic and may vary depending on their specific nature - so I did not even attempt to summarize).