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DNA isolation from plant nuclei by plasmid extraction kit


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#1 vietbio

vietbio

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Posted 13 October 2010 - 11:53 AM

Hi

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

either
+ transfer supernatant to Fermentas plasmid isolation column and wash and elute by warm H2O

or
+ 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?

#2 Ameya P

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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.

Good luck.... :)

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