I wanted to talk a little about the selection characteristics of Agencourt’s AMPure beads, a bead-reagent combination that purifies PCR reactions.
AMPure XP Manual or Automated Purification & Cleanup Nucleic acid purification and cleanup are mandatory for genomic applications including sequencing, qPCR/ddPCR/PCR, microarrays and other enzymatic reactions. 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. Agencourt AMPure XP beads This user bulletin includes updated instructions for performing step 7 of the Genome-Wide Human SNP 6.0 Nsp/Sty Assay. This document is intended for customers who are using the original SNP Assay 6.0 Kit. You may use this bulletin in conjunction with the quick reference cards (QRCs) to view relevant diagrams. Contained in the exact buffer at this step (71.5 µl; Step 1.2.5.). AMPure XP Beads can be used as well. If using AMPure XP Beads, allow the beads to warm to room temperature for at least 30 minutes before use. These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step.
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.
Ampure Xp Beads Manual
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.
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.
Beckman Coulter Ampure Xp
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.
Without any further ado, here are the results: