I isolate RNA from Gram-positive bacteria using Trizol reagent (Invitrogen).
I would like to know why the centrifugation speed should be reduced from 12000 g to 7500 g during the ethanol wash step? Would anything happen to the sample if I used the same speed of 12000 g throughout the purification?
The reduction in centrifugation speed during the ethanol wash step in RNA isolation using Trizol reagent/invitrogen is typically done to minimize the risk of RNA loss. The purpose of the ethanol wash is to remove salts and other contaminants from the RNA pellet. Higher centrifugation speeds can result in a more compact pellet but may also lead to co-precipitation of salts and other impurities.
Reducing the centrifugation speed to 7500 g allows for a gentler separation of the RNA pellet from the ethanol and residual contaminants. This helps to prevent the pellet from becoming overly dense, which could lead to RNA loss or decreased RNA purity. The lower speed is often sufficient to pellet the RNA while leaving behind unwanted substances in the supernatant.
Using the same high centrifugation speed throughout the purification may result in a tighter, more compact pellet, but it also increases the likelihood of losing RNA or retaining impurities in the pellet. The lower speed during the ethanol wash step is a precautionary measure to optimize RNA recovery and purity.
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