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Quiagen DNA extraction kit problem

DNA viscousity Quiagen DNA kits

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3 replies to this topic

#1 calobo

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Posted 10 January 2012 - 07:46 AM

Hello.

I´m 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

#2 mdfenko

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Posted 10 January 2012 - 09:40 AM

(i don't have the experience that you request, but...)

mucus-like glop is often caused by genomic dna or by carbohydrate (or both).

if it is caused by genomic dna then the glop can be eliminated by sonication.

ctab extraction (i know you tried) is usually used to avoid the glop caused by carbohydrate.

could it be that your solution is just too concentrated? you can try using less starting material or you can try dilution to reduce the viscosity.

there's also the possibility that your extraction conditions are causing some protein(s) to polymerize or aggregate.

Edited by mdfenko, 10 January 2012 - 09:42 AM.

talent does what it can
genius does what it must
i do what i get paid to do

#3 cbf88

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Posted 10 January 2012 - 02:09 PM

Chloroform phenol extraction

#4 Fredo

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Posted 31 January 2012 - 06:15 AM

I think that the viscosity of your CTAB DNA extraction is caused by the presence of loads of polysaccharides . these compounds stick to DNA especially if your sample was frozen...
Are you using PVP40 in your CTAB buffer? PVP helps to get rid of polysaccharides. You can also use activated charcoal to get rid of polysaccharides.
I'm working with grapewine which is species containing loads of polysacch. and polyphenol . After my CTAB extraction , I clean up my samples by using this type of collumn :http://www.mn-net.com/tabid/1452/default.aspx and it works quite well.
I manage to get 15 ug / g of fresh leaves. Then the DNA is ok for PCR or restriction !

Good luck

Edited by Fredo, 31 January 2012 - 07:48 AM.






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