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Inhibitory agents eluting with DNA from spin column extraction method

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#1 Micro



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Posted 04 August 2015 - 06:43 PM

I am using a Promega SV genomic DNA extraction kit with a modified protocol for extracting Gram positive bacteria. See link for details: www.sjsu.edu/people/peter.nemes/courses/Chem131A/s2/Genomic-DNA-Purification-Protocol.doc

I am finding that when I use the eluted DNA in qPCR the reaction is being inhibited. I suspect that a contaminant is being eluted with the DNA, so I did a NaAc/EtOH precipitation and then repeated the qPCR, and the samples produced the expected response. I’m reluctant to routinely use the NaAC/EtOH going forward, because I will be quantifying the DNA in the reaction and this additional step will increase error in the results.


My plan going forward is to dilute my DNA 1:100 to reduce the contaminant, but my curious mind want to actually find a solution.


Has anyone else experienced this problem?


Does anyone have any suggestions potential sources of contaminants from spin column DNA extractions?

Thanks for your help!

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