I extracted the DNA present in a commercial inoculum of mycorrhiza, where the carrier is mainly dextrose, with PowerSoil DNA Isolation Kit and I have problems with the subsequent amplification. I think it could be due to the high amount of dextrose. Any suggestion to eliminate the excess of sugars?
I don't have a direct answer to your question, however, I might have an idea how you can get your amplification to work. I work with DNA from marine environmental samples and there are known inhibitors present. Thus we have to dilute our sample DNA prior to the amplification. For example a 1:10 dilution of the DNA sample in H2O works well but we were never able to get an amplification from the undiluted DNA sample. I would give it a try it might solve your problem
A straightforward dilution as above may be the answer (though you might need to increase the number of cycles you amplify for).
Remember there are several other PCR parameters which should be investigated in the first instance as I have never heard of dextrose preventing PCR.
However, you could probably get rid of the dextrose by methanol precipitation; I'd try about 80% methanol (so 200ul sample in 800ul methanol).
I'm no chemist but I think the reason this method would work is because, although both act polar in water, individual dextrose molecules remain dissolved in the 20% water, whilst longer DNA sequences are forced to precipitate and form a pellet upon spinning.
Good luck and let us know how it goes!
Thank you for your answers. Since the main (and presumably the only) component of the carrier seems to be glucose/dextrose and the NanoDrop readings for 260/230 ratio were quite low (0.5-1.0), I was assuming that the glucose was somehow inhibiting my reactions, but as you say, maybe it is not the case (I found some information about carbohydrate inhibition of Taq polymerase...). Anyway, diluting the sample 1:10 gave a good result in most of the extractions so I will try to dilute even more in the ones that no positive result was obtained. My DNA yield is not very high (5-20 ng/uL) so I am afraid of diluting the samples too much.
Thanks again for your interesting feedback! :)
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