I've got samples of concentrated urine (0,5 ml) containing Schistosoma eggs, stored in 1 ml of ethanol. I understand that normally you perform a washing step with PBS to get rid of the ethanol before DNA isolation and subsequent RT-PCR. I accidentally took the unwashed samples and found out that there was no significant effect on the PCR results. So my question is: why is there a washing step? What kind of effect does ethanol have on DNA isolation?
I think the ethanol is there as a preservative of the dna....enzymes and bacteria etc cannot work in the alcoholic environment. The washing step is to get rid of the alcohol because it might interfere with the pcr reaction by changing the structure or precipitating the taq polymerase but if you only used a small volume of sample in a large volume pcr reaction then the pcr will work
Hi Dennis. Since ethanol forms interactions with the water molecules by the presence of hydroxyl groups, it decreases the number of water molecules available to hydrate the DNA. Ethanol also has a low dielectric constant which together makes the DNA less soluble and causes the DNA to aggregate with positive ions in the solution (which is why it is used to precipitate the DNA). And because I think soluble DNA is required for PCR reactions to make it available for polymerase, the washing step must be required. In addition, the washing step will also remove any low molecular weight contaminants if present any.
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