For the precipitation of DNA during extraction protocol, which is preferred, among isopropanol and isoamylalcohol and what is the underlying reason to select it ?
Hello Dear Bhavya G.
f you are precipitating small volumes of DNA, and you can fit the required amount of solvent into the sample tube, then ice cold ethanol is the preferred choice. You can chill it (some people use liquid nitrogen or –80°C to accelerate the precipitation) and precipitate more DNA without the salt contamination that would occur from chilling isopropanol. Afterwards you need to wash the pellet with 70% ethanol to remove salt.
Isopropanol use useful for precipitations where you have a large sample volume (e.g. the eluate you get after using a Qiagen plasmid Maxi Kit) because less solvent is needed, so you can fit the whole lot in the (15 ml) tube. But because salts are generally less soluble in isopropanol than in ethanol, they have more of a tendancy to co-precipitate with the DNA. So to lessen the chances of salt precipitation, isopropanol precipitations are carried our at room temperature with minimal incubation times. Once the DNA or RNA pellet is recovered from the isopropanol, you’ll want to wash it with cold 70% ethanol to remove excess salt and to exchange the isopropanol with the more volatile ethanol. It is ok to chill the isopropanol precipitated sample, if you are sure that it is not excessively salty.
Because DNA is less soluble in isopropanol, isopropanol allows precipitation of larger species and lower concentrations of nucleic acids than ethanol, especially if you incubate it cold and long. If you do this, just remember to wash the pellet several times in 70% ethanol after pelleting, to reduce the amount of salt you carry over.
Hello Dear Bhavya G.
f you are precipitating small volumes of DNA, and you can fit the required amount of solvent into the sample tube, then ice cold ethanol is the preferred choice. You can chill it (some people use liquid nitrogen or –80°C to accelerate the precipitation) and precipitate more DNA without the salt contamination that would occur from chilling isopropanol. Afterwards you need to wash the pellet with 70% ethanol to remove salt.
Isopropanol use useful for precipitations where you have a large sample volume (e.g. the eluate you get after using a Qiagen plasmid Maxi Kit) because less solvent is needed, so you can fit the whole lot in the (15 ml) tube. But because salts are generally less soluble in isopropanol than in ethanol, they have more of a tendancy to co-precipitate with the DNA. So to lessen the chances of salt precipitation, isopropanol precipitations are carried our at room temperature with minimal incubation times. Once the DNA or RNA pellet is recovered from the isopropanol, you’ll want to wash it with cold 70% ethanol to remove excess salt and to exchange the isopropanol with the more volatile ethanol. It is ok to chill the isopropanol precipitated sample, if you are sure that it is not excessively salty.
Because DNA is less soluble in isopropanol, isopropanol allows precipitation of larger species and lower concentrations of nucleic acids than ethanol, especially if you incubate it cold and long. If you do this, just remember to wash the pellet several times in 70% ethanol after pelleting, to reduce the amount of salt you carry over.
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