I wanted to talk a little about the selection characteristics of Agencourt's AMPure beads, a bead-reagent combination that purifies PCR reactions.
Requiring no centrifugation or filtration, AMPure XP can be easily used in manual and automated 96- or 384-well formats. Option A: Agencourt AMPure XP/SPRIselect Bead Cleanup 1. Vortex AMPure XP/SPRIselect Beads to resuspend. If your enriched sample volume x 2.8 from the previous step exceeds your tube volume, split into two tubes. Add 1.8X volume of resuspended AMPure XP or SPRIselect beads to the sample. Mix well by pipetting up and down at least 10 times. Beckman Coulter Life Sciences is taking actions in the best interests of our associates, customers, and business partners as we navigate the growing threats of the 2019 Novel Coronavirus disease (COVID-19). About This Manual 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. Consistently, 1% of sequencing reads from the Agencourt AMPure XP kit is smaller than 100 bp but nearly 5–10% of sequencing reads are smaller than 100 bp from column-based purification reagents (Additional file ). We tested whether purification reagents utilizing silica columns or SPRI may introduce any bias in ChIP-seq data.
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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.
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.
Beckman Coulter Ampure Xp
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.
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.
Beckman Ampure Xp Beads
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.
Beckman Coulter Ampure Xp
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.
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.
Beckman Ampure Xp Beads
Ampure Xp Beads Manual
Without any further ado, here are the results:
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