Dear all, I have used iron beads for DNA isolation using homogenizor. But, after adding the extraction buffer, the solution started black in colour. I applied Chloroform:Isoamyl alcohol (24:1) solution but not able to remove the colour. Although I got intact DNA band after resolving the DNA prep in 1% agarose gel. But, I doubt that the colouring substances may hinder the PCR amplification.
Kindly suggest me some option to tackle the above said problem.
Gopal Singh
I am not familiar with the use of iron beads for DNA extraction. Can you provide a URL for the method, please?
Hi,
Though I never used iron beads, but we also used to get coloured pellets while extracting DNA from soil samples. But we always got good amplification from the DNA. Maybe you should to try once. Just use less amount of template, in order to avoid incorporating too much PCR inhibitors.
Hope it helps.
Dear Geoff, I used common CTAB method for DNA isolation. Iron beads were used only to homogenize the leaf tissue instead of using liq. nirtogen. the rest of the protocol is same.
Thanks for your query.
My guess is that the black material is pulverised and/or oxidised iron. But you dont say if your DNA is black. If it is, then CETAB presumably precipitated the iron too. If you add Tris-EDTA buffer, to dissolve the DNA, can the black material be removed by centrifugation? Even if it can, the DNA solution will contain a high concentration of iron ions that may well interfere with PCR. But by now, do you know the result of PCR??
Dear Geoff, I have the same concern about presence of iron will hinder PCR amplification. This is why I want to remove iron contamination from my DNA prep.
Dear Paul, I am trying your suggestion. Hopefully it will short out my problem.
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