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calobo 01-10-2012 03:59 PM

Problems in DNA extraction using kit
 
Hello.

Im presently carryng out a molecular study with some plant species of the Family Lauracea (Persea mostly).

Presently, the DNA extraction is being carried out using Quiagen DNA extraction kit, with which I have had good results in the past with other plant species. Nevertheless, with several species of the Persea genus things are quite different.

I grind the plant material with liquid nitrogen and soon after I add the AP1 buffer (lyses buffer), a very viscous, mucus like substance results. If I carry on the protocol as recomended by Quiagen and add the AP2 buffer (precipitation buffer), centrifuge and after which I atempt to recover the supernatant, in several cases there is no supernatant to recovery. Just a block of a mucus in the tube.

Whenever any supernatant is recovered, it is translucent, although quite viscous. Carriyng out the extraction untill the end, in most cases there is no DNA obtained from the extraction, as evidenced by the absence of bands in a electrophoresis.

I have tried altering the procedure recomended by Quiagen in an atempt to obtain DNA, but untill now with little or no success. The alterations are mostly related to alterations in the volume of AP1 and/or AP2 used during the extraction.

Does anyone have similar experience in situations similar to this one and can you please give me some guidance on how to obtain DNA from plants that produce very viscous substances?

PS: I have tried extracting DNA from these species using CTAB. The very viscous substance also forms and in general the sucess of the DNA extraction in very low.

Thank you in advance for your possible interest and help in solving this issue.

Best wishes

Carlos Lobo


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