DNA isolation from plant nuclei by plasmid extraction kit - (Oct/13/2010 )
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?
vietbio on Wed Oct 13 19:53:11 2010 said:
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.