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Old 05-08-2014, 10:34 PM   #1
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

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Default Freshness of Ethanol used for washing AMPure beads

Most of Illumina library prep protocols which uses AMPure beads for clean up or size selection recommends using freshly prepared 70% or 80% Ethanol for wash step. Both works very well and I have not noticed any difference in performance of eluted libraries washed by either. I wonder how often or how fresh any one else prepares their wash Ethanol. Fresh prep in literature is justified by presumed or real changes in ethanol concentration by absorption of air moisture. Considering that two recommended wash solutions differs by 10% in Ethanol content does freshness really makes any difference.
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Old 05-09-2014, 12:57 AM   #2
Chipper
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It does not need to be made fresh. Possibly altered EtOH concentration affects washing but you will not lose DNA unless EtOH is below 40 % or so (I have not tested this myself but it can easily be done with a simple titration using a ladder).
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Old 05-19-2014, 12:53 PM   #3
kzarn
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I have recently spoken to a few technicians and post doc about this. Everyone I know makes fresh ethanol for AMPure bead clean ups or size selection - some make 73%, others 74%, 75%, etc. (exact number seems to just be personal preference).

Found this, supporting what Chipper said: "The original AMPure protocol recommended use of a 70% ethanol wash solution. This needed to be freshly made, as over time it becomes more dilute since it absorbs atmospheric water and ethanol evaporates. When you rinse with more dilute ethanol more DNA gets washed away. We have found that this can be avoided by using an 80% ethanol solution instead. This washes just as well but does not need to be freshly prepared each time."
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