MadSci Network: Molecular Biology
Query:

Re: why is there a difference in amount of DNA extracted

Date: Fri Apr 13 15:13:11 2001
Posted By: Sabine Heinhorst, Faculty, Chemistry and Biochemistry, The University of Southern Mississippi
Area of science: Molecular Biology
ID: 984365666.Mb
Message:

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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