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impurities after DNA extraction with promega kit!!!!!!&#


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6 replies to this topic

#1 lula

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Posted 10 November 2012 - 12:04 PM

hi
a student in my lab got from abroad with DNA samples extracted by promega kit as he say and the only paper from the lab he worked for is the information for kit shipment with a stament that the DNA extraction is to be applied in msystems???
the problem is that the samples were brown fullwith hb and debries i checked on agarose gel and nothing appperaed ,,,,i dont have a spectrophotometer in my lab
does any one know what is the problem??????????????????
i think my student said they used guanidium thiocyanate ?? is that possible in DNA extraction??

#2 bob1

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Posted 10 November 2012 - 12:27 PM

Guanidinium is common in DNA extractions, it is used to denature the proteins so that they can be removed from the DNA.

What organism are the samples extracted from ? - plants and fungi often have components that will result in a brown DNA solution, some bacteria will also do this.

#3 lula

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Posted 10 November 2012 - 12:50 PM

the samples are of human blood and the debries are haemoglobin ,,,,i know that each kit for extraction gives u clear lysate for DNA at the end not brown am i right

#4 bob1

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Posted 10 November 2012 - 01:00 PM

Oh, right - Hb, I just thought you had made a typo or something. Usually, blood extractions would be pretty clean, though it might depend on if they did a buffy coat or not, and how much blood they loaded into the extraction.

You could try to clean up the DNA by putting it through a salt precipitation and/or a phenol-chloroform extraction.

#5 lula

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Posted 11 November 2012 - 12:53 PM

actually thats a great idea i will try it thank u

#6 Trof

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Posted 20 November 2012 - 03:17 PM

If that brown thing in your DNA is heme contamination from a badly isolated blood (usually erythocytes are lysed and removed completely before lysing the leukocytes), you need to get completely rid of that before you try to use the DNA for any PCR method. Heme is a potent PCR inhibitor.
But if it is, even if you get rid of the color, the traces may still remain there. Adding BSA to the reaction should help to bind-out heme in that case.

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#7 pito

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Posted 21 November 2012 - 11:35 AM

If that brown thing in your DNA is heme contamination from a badly isolated blood (usually erythocytes are lysed and removed completely before lysing the leukocytes), you need to get completely rid of that before you try to use the DNA for any PCR method. Heme is a potent PCR inhibitor.
But if it is, even if you get rid of the color, the traces may still remain there. Adding BSA to the reaction should help to bind-out heme in that case.

I have always wondered: the BSA, would it really help that much?
BSA denaturates between 55-70°C, so how can it still work during PCR.

Or do you add the BSA prior and extract it, bound to the heme?

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