DNA isolation from plant nuclei by plasmid extraction kit
Posted 13 October 2010 - 11:53 AM
I have my plant nuclei in 0.5x PBS and want to isolate DNA from those. I borrowed some P2 and P3 from Fermentas plasmid extraction kit and tried to break plant nuclei simply the same as bacterial cells.
+ add 1 volume of P2, invert tubes several times, RT 10 min
+ add 1.5 volume of P3, invert tubes several times, RT 5 min
=> can found a lot of protein were precipitated
+ centrifuge 10.000xg 1min => collect supernatant
+ transfer supernatant to Fermentas plasmid isolation column and wash and elute by warm H2O
+ precipitate DNA by RT isopropanol, wash with EthOH 70%, resuspend pellet (can not be visible) in H2O
Unfortunately none of DNA can be found in those samples. What was wrong? Anybody have any idea?
Posted 13 October 2010 - 10:00 PM
precipitate DNA by RT isopropanol, wash with EthOH 70%, resuspend pellet (can not be visible) in H2O
I am sure that you have tested the presence of DNA, by running on a gel. If you could post a picture, so that we can see if there is atleast RNA somewhere.
It might be be useful to add 0.5ul - 1 ul of Glycogen to isopropanol to get a visible pellet.
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