DNA Isolation

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Boy spindling DNA Isolation of DNA from Strawberries and Raspberries
Works for onions also!
Duration = 40 minutes.
Girl with spindled DNA

 
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Note that these are fruits of highly composite flowers. The method works for both fresh and frozen berries.

Chemists have long wished for a reaction in which they could use the "tweezers method" - to reach into their pot of "soup" and grab just the molecules they want and leave all the others behind. This DNA isolation is just that - a tweezers method of purification. It is all because DNA is such a long molecule. You will eventually snag it by one of its ends. As you reel it in, it will snag other DNA molecules and they will come along. Soon on your rod you will have a large spindle of DNA. The following is for a class of 20 students:


Materials needed


  1. Put 100 ml of fresh strawberries into a high speed blender, and fill up to the top of the fruit with homogenizing reagent*. Do NOT turn on the blender. Plant cells are surrounded with cellulose walls that are easy to crush so that the cells' guts can spill out into the liquid. A mortar and pestle also work well. This reagent has three important ingredients. NaCl is to keep the DNA in its double helix form, otherwise it would denature and become "ssDNA." The EDTA is to bind up Mg++, which is a necessary cofactor of DNase, which can "eat" your desired product - the DNA. And the anionic detergent, sodium dodecylsulfate (aka "SDS") plays two roles: one is as a detergent to discombobulate lipid layers and make the membranes dissolve, and the other is to bind with the positive charges of the chromosomal proteins to release the DNA into solution..

  2. Put the top on the blender! Briefly blend with three pushes of the button. That means: "errrr, errrr, errrr." Blending is very effective in cell disruption as it makes huge shear forces that rip molecules from one another. But the blending must be very brief when isolating DNA, because the shear forces rapidly break the DNA into shorter and shorter lengths - just like stirring a pot of spagetti causes them to break into shorter and shorter noodles.

  3. Pour the blender's contents into a metal sauce pan and place it in waterbath at 55-60c for 15 minutes to lyse the cells and release the DNA. Once the contents reach 55C, start timing the 15 minutes. Occasionally slowly stir or swirl. (Do not go over 60c else the DNA will denature.) (You can try heating the pan directly with CONSTANT stirring. Two reasons for this temperature treatment: one is to partialy denature DNase, which is your big enemy, and the other is to soften up lipid membranes and to help the detergent molecules to slip in among the fluid mosaic molecules and cause the membrane sto fall apart and dissolve. A metal pan is suggested to speed the project as metal conducts heat much more rapidly than does either glass or plastic.

  4. Place the pan in ice water and swirl until the contents are below 10C. Cell components and excess detergent will precipitate. You want to precipitate the SDS because doing so will concommitantly also bring down the chromosomal proteins.

  5. Filter the thick puree through a #6 Melitta coffee filter or a paper towel cupped into a large beaker or clear plastic drinking cup.

  6. Distribute the filtrate into cups so that each student has about 1 cm (half inch) of liquid.

  7. DNA precipitation: gently and slowly pour 3 volumes of ice-cold 70% ethanol (rubbing alcohol) or isopropanol down the side of the beaker overlaying the aqueous DNA solution.

  8. Allow the beaker with its two layers to sit quietly for 3 minutes. A white thread-like substance (DNA) whisps up from the interface (Figure A)

  9. Very slowly rotate (not stir) a drinking straw (roughened by sandpaper) or a stirring rod in the mixture. Rotate the straw or rod about 1 turn every 4 seconds. DNA should collect as a fibrous spindle on the rod (Figure B). Against the side of the beaker, gently wring out of excess liquid from time to time (Figure C). Keep spinning and extracting the DNA until no more spindles onto your glass rod. More violently swirl or stir the solutions to get more of the ethanol into the aqueous layer, and allow to sit quietly for several minutes: more fibrous DNA will come out to be spindled on the rod. This will probably be the most unusual precipitation that you ever do in your life because it deals with extremely long molecules. The textile industry does this sort of thing all the time as they inject streams of liquid polymer into a precipitator liquid. The polymer streams become solid, and are reeled onto spindles at the other end of the precipitator vat.

  10. You may place your spindle into 95% ethanol for saving, or

  11. You may redissolve it in more homogenizing reagent or in "SSC" (0.9% NaCl, 0.1% Na3Citrate) for further studies such as viscosity measurements, or making a melting curve and seeing the hyperchromic effect - further evidence of double helix. Again remember that your big enemy is DNase - even from dust. It is a wise DNA-isolater who makes sure that EDTA or citrate are present as Mg++ chelators (ask your teacher about that word!)

  12. CLEAN UP TIME! You must wash your glassware with soap and water.

  13. DISCUSSION TIME: You have gone through a lot of motions which the directions led you to believe that you were isolating DNA. Suppose you were 25 years old and you had a large source of funding. What are various ways you could employ to show that you have in fact isolated dsDNA? That it is double-stranded, and that it is DNA? (Hint: what trick did the Nobel laureates Avery, MacCleod and McCarty use to show that DNA was indeed the genetic material in cells? Or what trick did Nobel laureates Meselson and Stahl use to show the same thing?)

  14. Modern molecular biologist researchers isolate DNA from various sources daily. However, most of them have never seen DNA like you have. That is because these scientists work with such small amounts of it that it is too little to see. Thus most of these people have never had the thrill of doing what you have just done. If this lab really excited you, make sure to mention the wonder it held for you in your college applications.

  15. Now that you have a spindle of DNA, what could you do with it? Suppose you had isolated pure strawberry DNA, and your friend had isolated some from a strain of strawberries that were frost resistant, or resistant to insect infestation. How would you go about isolating the frost or insect resistance genes? Once you have isolated them, how might you do a little genetic engineering to get those genes into another plant?


* Three component homogenizing reagent: 0.9% NaCl, 0.3% EDTA (ethylenediamine tetraacetic acid), 1% SDS . BEWARE: SDS dust can cause debilitating coughing fits! Weigh it out in a hood. 0.15M trisodium citrate may substitute for the EDTA. (Alternatively, to a large flask or bottle add 970 ml of water, then dissolve in 9 gms NaCl (0.9% NaCl), and add 30 ml of a shampoo that specifically contains lauryl sulfate or dodecylsulfate, and EDTA - Eckerd's Extra Mild Shampoo works very well.**)

** As an example for what to look for in other shampoos, here is what is in the Eckerd product, and the bold ingredients are the needed ones: water, ammonium lauryl sulfate, cocamide DEA, cocamidopropyl betaine, glycerin, citric acid [or citrate], methylparaben, propylparaben, diazolidinyl urea, disodium EDTA, fragrance.

DNA NOW is a similar commercial preparation. A demonstration size kit costs $10. BIOGENTEX / P. O. Box 74 / Seabrook, TX 77586 / Cat. #SP-101


Historical note: Gierer, A. and G. Schramm. 1956. Infectivity of Ribonucleic Acid from Tobacco Mosaic Virus. Nature 177: 702-703.




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