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chloroform/Qiagen Dneasy plant mini kit


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#1 aces

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Posted 07 August 2011 - 06:01 PM

Hi all,

Has anyone experienced DNA extraction using CTAB or SDS/chloroform in combination with Qiagen DNeasy plant minikit? I have to extract DNA from large volume of samples (ca. 3 g each) which contains high secondary metabolites, especially starches. There is a limitation on sample loaded for the Qiagen plant minikit so that I am thinking to use chloroform first before moving onto the kit. Anyone has suggestions/tricks?

Thank heaps in advance!

#2 David C H

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Posted 09 August 2011 - 06:01 AM

Qiagen may have guidelines for doing this.

After chloroform extraction, for 500ul of recovered aqueous solution, add about 20ul of 3M KOAc, pH 5.2 (recipe in Maniatis). Add 1 volumes EtOH or 0.7 volumes isopropanol, mix by pipette and add to the Qiagen column and proceed with the Qiagen protocol.

CTAB buffers are usually at a basic pH (if the lysis buffer and chloroform are acidic, DNA may precipitate as part of the solid interphase layer during the organic extraction -- this is how TriZol and similar methods seperate DNA from RNA isolations). Adding the acidic KOAc should drop the pH below 7.4; nucleic acids, especially DNA will not bind to the column at basic pH.

3g is a lot of tissue -- older protocols with higher starting volumes usually aren't written for more than 1g. Even with CTAB/chloroform extraction, if you overload your buffer with too much tissue you will end still up with less DNA and it will be less pure (more polysaccharide content). You will be better off finding a CTAB protocol or, if you want to use a kit, a maxi kit (Qiagen makes a plant DNA maxi kit).




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