During nucleic acid extraction after precipitation with 100% ethanol, we wash the pellet with 70% ethanol to remove excess salts from the pellet.
But during these washes, nucleic acid can also solubilise in some of the 30% of the water.
How much of the nucleic acid is lost? Has someone done any estimation?
Thanks
Hello Muhammad, how are you? So, I think nobody did that kind of estimation. You will have some lost of DNA or RNA during washing process with Isopropanol or ethanol for sure. However, generally that lost is insignificant when you will perform your analysis. If you working with DNA, with concentrations about 50 ng/ul you will get good results in PCR analysis. With RNA to synthesize cDNA, we usually use 1ug of purified RNA in the most kits for reverse transcriptase. If you working with low quantities of cells or your sample are too small that will yield lower quantities of DNA or RNA, I recommend you use extraction kits to avoid that lost.
Precipitated nucleic acids are not soluble in 70% ethanol. However, the nucleic acid pellets are easily lost during the washing procedure, especially if they come off from the bottom of the tube. Thus, greatest care is needed when sucking off the 70% ethanol supernatant. A good way to make the pellet more visible and to avoid losses is to add a few ug of molecular biology grade glycogen as a carrier.
Pierre Béguin Thanks. how to remove glycogen from DNA/RNA if need to send it for NGS? will it affect the DNA/RNA quality?
No,glycogen molecular biology grade doesn’t interfere with most enzymes acting on DNA (restriction enzymes, polymerases, nucleases, ligases, kinases etc.). It doesn’t absorb at 260 or 280 nm, so it doesn’t interfere with UV qualification of nucleic acids either. Thus, no need to eliminate it.
Usually, If the pellet is centrifuged properly, it should settle down at the bottom firmly. It is advisable to spin the samples a bit longer, prior to the washing step. However, while following the washing procedure, one shouldn't tap it hard after the addition of washing reagent which may cause the pellet to come loose while decanting the reagent (70% Ethanol).
Bappa Karmakar so you are saying that during the washing step, the pellet should not be completely resuspended in 70% ethanol. Am I right?
Muhammad Ehsaan
What I mean to say is that once you pour 70% ethanol, tapping gently at the bottom of Eppendorf tubes will make sure that the pellet is soaked completely. But not tapping to an extent, where the pellet come loose along with the reagent.
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