SPRI Bead Size Selection: Ethanol concentration effect

[sk8bro]

I am curious about why the Ampure XP protocol calls for 70% Ethanol, while their SPRI Select (supposedly the same as Ampure XP with extra QC) calls for 85% Ethanol.

Has anyone used 70% with the SPRI Select product from Beckman?

Has anyone done an Ethanol concentration titration experiment to see if there is any effect on size selection?

I've read that lower than 70% Ethanol impacts yield, while higher than 70% Ethanol concentration results in inefficient washing away of smaller molecules. What is the mechanism? Anypne have any data?

Reference from Nature Protocols: http://www.gmo-qpcr-analysis.com/tan...ocols-2010.pdf

Thanks for your advice!

[brofu]

Lowering the ethanol % in the wash steps will lower the overall yield without affecting the size distribution (as opposed to SPRI beads, where decreasing the amount of beads increases the insert size). If your yields are high enough you might be willing to sacrifice some good stuff to get rid of noise. If you've already tried decreasing the SPRI ratio as far as it could go and you still need more stringency then lowering the ethanol % could help.

[ScienceGrrl]

For all SPRI products (AMPureXP, SPRIselect, etc) if you go lower than 68% EtOH, you will cause the nucleic acid to elute off the beads. For manual prep, there is usually not an issue so earlier protocols state to use 70%. As Beckman did more and more automation with the SPRI beads, they upped the concentration of the EtOH so it could sit on deck of the robot without dropping below the 68% threshold and allow for the errors that would sometimes happen if a user prepared the EtOH dilution incorrectly or used older prepared batch of EtOH. Going above 85% is not suggested because you decrease your yield as you are more likely to overdry the beads and cause the nucleic acids to be unable to elue properly.