MadSci Network: Molecular Biology |
Dear Dorian, Sounds like you are involved in a very exciting research project. I cannot tell you the exact reasons for your variable DNA yields without having performed these and further control experiments myself, but here are a few educated guesses: 1. As you already surmised, different plants and plant tissues have different water content, so that on a per gram basis, you would obtain different amounts of DNA. Furthermore, different plants also have different DNA contents. The wheat variety that was processed into the wheat germ you used may have been polyploid (= have had more than 2 copies per chromosome), so that its DNA content per cell would have been higher than that of a diploid (2 chromosome copies per cell) plant. 2. Plants contain a variety of nucleases. These are enzymes that degrade the long DNA strands into short pieces that do not precipitate very well with alcohol and certainly do not spool onto a glass rod. Most of these nucleases can be inhibited by adding a little EDTA (that's short for ethylene diamine tetraacetic acid), which binds the metal ions that are needed by the nucleases. The different plant tissues you extracted may contain varying amounts of these nucleases. 3. I assume that you used the same DNA extraction protocol for all plant species and tissues. Those experimental conditions may not have been optimal for all plants and plant tissues. It is very difficult to extract DNA from many plants/tissues because they contain interfering components (e.g. carbohydrates, phenolic compounds), whereas other plants/tissues yield larger amounts of high quality DNA. You might want to experiment a little more and vary, for example, the amount of liquid in which you suspend your plant tissue, the amount of detergent you use to disrupt the plant cells, and the amount of salt you use. Does your protocol call for meat tenderizer? Meat tenderizer contains enzymes that degrade proteins. In some cases, that helps to release the DNA. I would not advise to use warm alcohol to precipitate the DNA, however. The colder the ethanol, the better the DNA will precipitate. Hope these tips will help. Without specialized equipment, determining the number of cells per plant sample would be very difficult. However, you can determine the plant's water content by weighing before (= wet weight) and after (= dry weight) dehydating in a warm oven or in a fruit dehydrator. Make sure that you weigh the plants several times during the dehydration process until their weight remains constant. At that point, all the water is lost. The difference between wet and dry weight is the weight of the water the plant contained. Good luck and lots of fun with your experiments! Sabine Heinhorst
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