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DNA extraction methods


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3 replies to this topic

#1 Patrick.T

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Posted 18 February 2009 - 02:26 PM

Actually, i have done an Genomic DNA extraction by using PCI DNA extraction method (using the most ancient protocol) . However, i cant obtain any precipitation of DNA even runing the agarose gel electrophoresis there is no band produced..... What are the possible mistake i had done during the experiment?
:o

Edited by Patrick.T, 18 February 2009 - 02:28 PM.


#2 bob1

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Posted 18 February 2009 - 03:15 PM

Are these questions homework? They sure sound like they are. Also, we can't easily help you unless you give a summary of the protocol including things like centrifuge speeds (RCF preferably), so that we can have some idea of where you might be going wrong.

#3 perneseblue

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Posted 18 February 2009 - 03:24 PM

how much material are working with? (Might it be possible that the reason you see nothing is because the quantity of DNA extracted is too low to detect by gel)

And what type of tissue is the DNA extracted from? Is there trouble homogenising the tissue? (Might it be possible that DNA fails to extract because it is retained in the tissue)

And when you say "agarose gel electrophoresis produces no band", is it an empty, clear, spotless gel. Or do you see a smear? Could the DNA be degrading.

Does your phenol/chloroform DNA extraction protocol use protenase? If so how long do you incubate at 55C? Over exposure to 55C will fragment the genomic DNA. Depending on use, this can be acceptable.

Could there be a mistake when making buffers? Do the buffer contain enough EDTA? Right pH. Has the phenol been equilibrated to pH 8?
May your PCR products be long, your protocols short and your boss on holiday

#4 mdfenko

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Posted 20 February 2009 - 07:12 AM

what is the concentration of the agarose in the gel? genomic dna stays near the top if it is intact and may not migrate at all if the agarose concentration is too high.
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