Purificação de PCR

O sistema AMPure XP da Agencourt é um sistema de purificação de PCR altamente eficiente e facilmente automatizado que fornece DNA de qualidade superior sem salinidade. Sem necessitar de centrifugação ou de filtragem, o Agencourt AMPure XP pode ser facilmente utilizado em formatos manuais e automáticos de 96 ou 384 poços.

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Overview

The AMPure XP bead-based reagent is intended for performing DNA cleanup steps in different genomic applications like sequencing, qPCR/ddPCR/PCR, microarrays, and other enzymatic reactions. It is powered by proven SPRI technology, which uses paramagnetic beads to selectively bind nucleic acids by type and size.

Works with DNA

High recovery of amplicons, greater than 100 bp

Efficient removal of unincorporated dNTPs, primers, primer dimers, salts and other contaminants

Predictable and consistent size selection

Maximizing recovery, consistency, and speed to facilitate the entire NGS workflow, the AMPure XP reagent meets the stringent needs of today’s genomic applications and minimizes the risk of losing important genetic information. That's why it's suggested in over 200 NGS library preparation kits often as the sole cleanup option, including those from the industry's most trusted sequencing companies like Illumina, Oxford Nanopore Technologies, PacBio and Thermo Scientific.

Specifications

Nucleic Acid Input PCR products, fragmented DNA
Output DNA
Recovery AMPure XP reagent was developed for the purification of PCR products 100bp<. Normal recovery ranges from 60-90%, and is typically between 70-90%.
Bead Ratio 1.8X for cleanup.Learn more →
Format Liquid
Volumes 5 mL, 60 mL, or 450 mL
Applications PCR Purification, DNA Cleanup, NGS Cleanup
Processing mode Automated or manual
Technology SPRI paramagnetic bead-based technology
Storage 4 °C

Data and Performance

Below are Agilent TapeStation system traces of sheared gDNA purified using the AMPure XP reagent, which shows that AMPure XP reagent provides predictable performance while maintaining efficient recovery - proven by the relative differences between the reference and 1.8X ratio.

AMPure XP Beads Size Selection Chart - Beckman Coulter life Sciences
CHART COLOR Bead/Sample Ratio
PINK Input/Reference
RED 1.8X
BLUE 0.9X
GREEN 0.7X
AQUA 0.6X

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Don't Lose Critical Data

When so much more has been invested in your research, AMPure XP reagent is the only choice for purification and cleanup steps. Loss of yield during this critical step leads to loss of discovery in your research. With the AMPure XP reagent, users can retain up to 34% more genetic information compared to other cleanup reagents.

Investing in AMPure XP Beads for Cleanup and Size Selection Retains Up to 34% of Genetic Information

The infographic shows relative costs of the different steps required to perform various NGS applications. Steps include extraction, library construction, library enrichment, cleanup and sequencing. Costs were calculated based on average list price of commercially available kits and reagents in 2017. Cleanup efficiencies were calculated by determining the total DNA yield by Picogreen Assay after performing a cleanup procedure on a known amount of DNA. The percent yield relative to AMPure XP reagent performance was then used to calculate the impact of efficiency on various commercially available library construction methods and a change in purification reagent.

Workflows and Protocol

AMPure XP Manual Workflow

AMPure XP Beads - Manual Workflow - Beckman Coulter Life Sciences
  1. Bind DNA to magnetic beads
  2. Separate beads from contaminants
  3. Wash magnetic beads with 70% ethanol to remove contaminants
  4. Elute DNA from magnetic beads
  5. Transfer to new plate

  

AMPure XP bead-based reagent can be performed either manually or automated on a liquid handling system depending on batch size or overall throughput.

In the table you can see estimated hands-on time and total time, in minutes, required to perform 48, 96, and 4 x 96 AMPure XP cleanups.

To learn more about automation of the AMPure XP PCR Purification system on the Biomek i5 Multichannel 96 Genomics Workstation, read our application note.

Read the Application Note

Manual Workflow Automated Workflow
Batch Size 48 Hands-on Time 25 5
Total Time 25 22
96 Hands-on Time 30 5
Total Time 30 22
4 x 96 Hands-on time NR 10
Total Time NR 46

NR = Not Recommended

Resources

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Data Sheet

 

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Application Note

 

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Customer Spotlight

 

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Application Note

 

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Frequently Asked Questions

The reagent will bind DNA and RNA; however, the RNAClean XP reagent is manufactured under RNase-free conditions and is QC tested to be RNase free whereas the AMPure XP reagent is not.

The AMPure XP reagent is manufactured and tested for the storage temperature indicated on the bottle and we can guarantee performance only at that temperature.

Find Beckman Coulter Life Sciences AMPure XP technical documents here.

Although size selection protocols developed by other organizations do exist, we cannot guarantee performance or support this application. We suggest using the SPRIselect reagent, as it is developed specifically for size selection purposes. Want to learn more about the difference between the AMPure XP and SPRIselect reagents? Visit this page.

No. Any beads carried over to the final destination plate are inert in downstream enzymatic reactions.

Yes, but the recovery will decrease with a decrease in elution volume. In addition, if the beads cannot reach the magnet during the separation it may be necessary to leave a portion of the eluate behind if bead carryover must be avoided. Be sure to resuspend the beads fully in the elution buffer.

The binding capacity of the beads is so high that they cannot be exceeded with any sample type. It is possible to bind at least 7 μg of nucleic acid to 1 μL AMPure XP reagent, however the sample will become so viscous that it is difficult to pipette the sample.

The recovery depends on the fragment size, sample volume, sample concentration, elution volume and some other factors. Under ideal conditions recovery can be up to 90%, but under sub-optimal it can be as low as 60%.

Our beads rely upon a multifactorial equilibrium chemistry for binding, washing, and elution. Binding buffer components facilitate nucleic acid immobilization at functional-group-rich binding sites on the beads and the wash steps preserve this delicate equilibrium while solubilizing contaminants to allow for their removal. Elution disrupts this balance and re-solubilizes nucleic acids to allow for their separation from the beads.
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A dry step can be incorporated after the removal of the last ethanol wash and before the addition of the elution buffer. For purification of very large fragments, such as gDNA, a dry step is not recommended because recovery would be decreased. Fragments up to 40 kb have been known to tolerate a dry step.

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Citations

AMPure XP reagents have been cited in over 52,000 publications on Google Scholar and referenced in articles in Science, Nature, and PNAS. Here are two featured articles:

Cleanup barcoded PCR reactions during library preparation
Greenwald WW et al. Subtle changes in chromatin loop contact propensity are associated with differential gene regulation and expression. Nat Commun. 2019 Mar 5;10(1):1054. doi: 10.1038/s41467-019-08940-5.
Cleanup cDNA
Peffers MJ et al. Transcriptomic profiling of cartilage ageing. Genom Data. 2014 Mar 19;2:27-8. doi: 10.1016/j.gdata.2014.03.001.

Documentos Técnicos

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Products and demonstrated applications are not intended or validated for use in diagnostic procedures.