I wanted to talk a little about the selection characteristics of Agencourt’s AMPure beads, a bead-reagent combination that purifies PCR reactions. Blue bird ikimono gakari download.
This stuff is incredible in terms of simplicity, efficiency, and high-throughput compatibility. I have a sneaking suspicion that AMPure, not unlike fire to Prometheus, was handed down from the gods to benefit humanity. You just dunk it into your sample, slosh it around, stick it to a magnet, wash, wash again, and elute in your favorite buffer. No muss, no fuss.
- The information in this manual is organized as follows: Agencourt AMPure XP PCR Purification Provides an overview of the Agencourt AMPure XP PCR.purification process, materials such as consumables and reagents needed, and the procedure for either a 96 well format or a 384 well.
- The Agencourt AMPure XP system is a highly efficient, easily automated PCR purification system that delivers superior quality DNA with no salt carryover. Requiring no centrifugation or filtration, Agencourt AMPure XP can be easily used in manual and automated 96- or 384-well formats.
- AGENCOURT® RNACLEAN XP® Protocol 001298v001 Page 2 of 2 For questions regarding this protocol, call Technical Support at Agencourt 1-800-773-9186 Agencourt Bioscience Corporation, A Beckman Coulter Company 800-361-7780 978-867-2600 500 Cummings Center, Suite 2450 Beverly, Massachusetts 01915 www.agencourt.com.
We were wondering, though, about its selection process. What size fragments are selected by the AMPure beads, specifically at which ratio of beads to sample? So, like diligent scientists, we rolled up the sleeves of our labcoats and… read the protocol.
AMPure XP clean-ups. AMPure XP can be performed either manually or automated on a liquid handling system. Difference in time between manual and automation is indicated. NR = Not Recommended For use in manual or automated methods based on batch size or overall throughput. Size Select the small RNA library using AMPure XP beads after using column purification. To the purified PCR reaction (25 μl), add 32.5 μl (1.3X) of resuspended AMPure XP beads and mix well on a vortex mixer or by pipetting up and down at least 10 times. Incubate for 5 minutes at room temperature.
The protocol recommends washing your sample in a 1.8:1 ratio of beads to sample, although it says that fragments less than 100bp will be omitted at this ratio, it doesn’t say which sized fragments will be selected. We found this remarkably helpful technical bulletin, which describes calibrating each batch of AMPure beads with various ratios of DNA ladder.
Ampure Xp Protocol
So I did our very own calibration with AMPure beads using Fermentas’s GeneRuler™ Low Range DNA Ladder (25-700 bp). I added 30ul ladder to various concentrations of AMPure beads according to Agencourt’s instructions.
(Actually, if you’re looking for good AMPure instructions, I recommend looking at Illumina’s TruSeq™ Sample Preparation Guide. Honestly, their instructions are more comprehensive than Agencourt’s, and easier to read.) After purifying each sample, I bookended the various AMPure:ladder ratios with 10ul non-purified ladder on a 2% TBE gel for easy comparison.
Ampure Xp Manual
Without any further ado, here are the results: