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Further DNA purification steps needed


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#1 Nebulus

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Posted 06 October 2009 - 02:14 PM

Hello. I have been using Qiagen DNAeasy Blood and Tissue Kits to extract prey DNA from the guts of their predators. There seems to be inhibitory substances present in my DNA samples even after using this extraction method. I ran tests by placing DNA solutions testing positive for prey remains with those that tested negative and the negative solutions always caused the positive solutions to become negative.

I optimized my PCR reactions using 25ng/ul of my prey DNA, which were diluted from stocks ranging from 50-120ng/ul. After DNA extraction from my predator's guts DNA concentrations range from 450-600ng/ul. I cannot dilute these solutions or risk not detecting prey DNA when predation occurred. I tried adding different %v/v of BSA to no avail. I know Qiagen also makes DNA stool extraction kits that contain InhibitEX tablets which are supposed to bind to inhibitors found in fecal matter, but I don't know what inhibitors are present.

Are there any additional DNA purification steps I could try after using Qiagen kits? Im not sure exactly what inhibitors are present. My predators feed on the phloem tissues of trees so there could be complex polysaccharides, and since I'm using the digestive tracts of my predators there could be inhibitors found there as well.

Any advice is greatly appreciated.

#2 gebirgsziege

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Posted 06 October 2009 - 10:47 PM

For the plant polysaccarides: try to add some PVP (Polyvinylpyrrolidone) to the first extraction buffer; app. 0.5 - 2% work usually fine.

Or to clean up the ready extracts: try PVPP (Polyvinylpolypyrrolidone) spin columns....I can pm you a paper with the method if you are interested.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#3 Nebulus

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Posted 07 October 2009 - 02:20 PM

Thank you very much. I will try both methods and let you know if they worked.

#4 Nebulus

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Posted 08 October 2009 - 09:45 AM

Quick question...

From my reading it seems PVP is mainly used in CTAB extraction methods. Will it be ok if I add it to my buffer ATL in my DNAeasy kit protocol?

Also, Ive seen 2 methods for loading columns with PVPP. One way is to dissolve the PVPP in tris-EDTA and add 2ml to the spin column then spin it down to bind it to the membrane. The other method just packs the column with about 22-30mm of PVPP powder and you pass the DNA solution through that.

Do you have any preferences?

Thanks

#5 gebirgsziege

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Posted 09 October 2009 - 02:44 AM

for PVP: I used it with CTAB and SDS Protocols with the same effect....so maybe you should just give it a try?

For PVPP: I usually add suspension in TE (app. 10% vol) to a 0.5mL tube with a small hole in the bottom, spin dry (low speed) and then add the DNA extract, spin again (like 1000 g, 2 min)....catch in a 1.5ml tube....finshed.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)




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