Does anyone have an idea of how much plant tissue (Leaves grounded in liquid nitrogen) should be processed in order to get at least 1 gram of DNA by classical extraction methods (CTAB)?
Hi, Annalisa,
generally, one can got up to 0,2 µg of the DNA pre mg of the tissue. If you have use old leaves with large cells, the amount are less.
I guess you will need at least 100 g of the tissue to get 1mg.
Goog luck!
Hi! Yes the leaves were quite old..I used 400 milligram and got moreless 150 micrograms then yes I think I have to use at least 100-200 grams of this material...
if you take old leaves, you need to crush it more and have to take more leaf sample as said above ,repeat the addition of Chloroform : Iso Amyl Alcohol (24:1) once more, sum up the upper aqueous phase and add Ammonium Acetate, ethanol accordingly. After adding ethanol invert tubes very gently so that the total precipitate will be altogether.
thank you all. yes in fact I have tried with more material (800 mg of crushed Leaves) and I am doing the chloroform extraction twice. so I get a cleaner , but however dark green DNA pellet. The amount until now is moreless 2-3 ml of 200 ng/ul of DNA solution...not so bad but with a quite low A260/A280 ratio....1.3-1.4
Follow the method on Plant Methods 8:26. I routinely used freeze-dried leaf samples from 20 to over 100 mg. I obtain 50 to 200 micro gram of DNA. You can use large preps to obtain mg of pure DNA
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