This precipitation can be used to concentrate small nucleic acids from dilute solutions such as the Lower Running Buffer after flashPAGE fractionation. When less than 2 μg nucleic acid is loaded onto the flashPAGE Fractionator, we recommend overnight sodium acetate/ethanol precipitation with a carrier such as linear acrylamide or glycogen for maximum nucleic acid recovery from the Lower Running Buffer. We do not recommend using glycogen as a carrier for samples that will be used for microarray analysis.
- Add 2 μl carrier (e.g. linear acrylamide Cat #9520 or glycogen Cat #9510) to the nucleic acid solution and mix well.
- Add 1:10 volume of 3 M sodium acetate and mix thoroughly; for 230 μl of Lower Running Buffer, this will be 23 μl of 3 M sodium acetate.
- Add 4 volumes of 100% ethanol; i.e. 1 ml 100% ethanol for 230 μl Lower Running Buffer plus 23 μl 3 M sodium acetate.
- Mix thoroughly and incubate at –20° C overnight (16 hr).
- We found that overnight incubation is important for maximizing recovery in this precipitation. (For example hr incubations resulted in significantly lower yield than overnight incubations).
- Microcentrifuge at top speed for 30 min at 4° C or room temp.
- Carefully remove and discard the supernatant.
- Wash the pellet by adding 500 μl 80% cold ethanol. Microcentrifuge at top speed for 10 min at 4°C or room temp, and carefully remove and discard the 80% ethanol.
- Repeat the 80% ethanol wash if needed to remove the large salt pellet from the sample.
- To remove the last traces of ethanol, quickly re-spin the tube, and aspirate any residual fluid with a very fine tipped pipette or syringe needle. Air dry the pellet.