I accidently added five fold more chloroform in the initial steps of RNA isolation. What are the consequences of this? Also, if i add more isopropanol to compensate this effect, how both chloroform and isopropanol are related ?
I assume you are using trizol or a similar reagent? Adding excess chloroform will alter the ratio of organic to aqueous substances in your extraction mixture and there's a high chance that molecules will partition inappropriately. Adding more isopropanol at a later step will not compensate for this effect at all. This extraction can be rescued by adding an excess four volumes of trizol (so that the ratio of trizol to chloroform is correct), vortexing, incubating for 5 minutes, and then proceeding with phase separation. You might need to transfer the whole reaction into a larger container like a Falcon tube, then do the mixing and incubation, then aliquot it into smaller tubes for phase separation spin. At the precipitation step, again, measure the volume of aqueous phase (it will be approximately 5 times as much) and add the appropriate amount of ethanol or isopropanol to make sure the RATIO is correct - it's the ratio rather than any absolute amount that matters, trizol reactions can be linearly scaled up and down. After precipitation steps, you can do pellet washes with the normal volume of 70% ethanol. If you had to split your sample over multiple tubes, try to elute each tube in a small volume and then pool them all together. You will likely get less sample (because precipitating RNA from a more dilute solution is less efficient) but it should be of similar quality and purity to your others and biologically comparable.
I will also note that if you are using Trizol LS specifically (or another liquid-specific reagent), you will also need to add extra water - 4 volumes compared to your original sample.
I recommend to start new RNA extraction and get rid of the previous. Because if you are going to continue rescuing your extraction I assume you will face a complicated issues with no result guarantee!!
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