Magnetic Bead Based Nucleic Acid Cleanup...2018/03/13 · nucleic acids • Paramagnetic Beads − Silica beads: use anion exchange principles for nucleic acid isolation − CleanNA

Magnetic Bead Based Nucleic Acid Cleanup

CleanNA is a registered trademark of GC biotech [email protected]



Nucleic Acid purification Methods• Traditional Methods

− Organic Solvents: Phenol/Chlorophorm, Cescium Chloride− Alcohol Precipitation: Ethanol, Isopropanol

• Anion Exchange− Charged resins used (commonly silica/DEAE) to bind nucleic acids

• Filtration− Gel (commonly Sephadex G50) or membrane formats− separation can be based either on size exclusion or preferential binding of filter to

nucleic acids

• Paramagnetic Beads− Silica beads: use anion exchange principles for nucleic acid isolation− CleanNA beads (carboxylated beads): binding buffers control size based binding of

nucleic acids onto beads


Magnetic Bead Based Technology

• Charged Magnetic Beads• Numerous binding options available with salts, alcohol group and detergents

优点： • High binding capacity , greater sensitivity • Best for automation, high throughput processing, no centrifugation

Bind Separation Wash Elution Transfer


CleanNA Advantages

10 min.0 min.

竞争对手产品

10 min.0 min.

CleanNA 磁珠

• Paramagnetic Microparticles Easily automated; no centrifugation or filtration

• Uniform Bead Size Highly reproducible results

• Negatively Charged Surface Aqueous elutions using no chaotropic salts

• Non-styrene Polymer Surface Low non-specific binding

• High surface area to mass ration Increased binding capacity

• Fast Magnetic Response Time Rapid purifications

• Size Optimized Particles Low settling rates for automation applications


How do CleanNA Products Work

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How do CleanNA Products Work

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EtOH

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CleanNA vs Silica purifications

• Non-specific binding

• Specific Elution

• Non-exclusive

Silica Bead-BasedPurifications CleanNA

• Specific binding

• Non-specific elution

• Exclusive to CleanNA

CleanNA versus silica-based beads • lower nonspecific binding• higher yield• cleaner preparation• more cost effective• unique & proprietary


CleanNA Reagents for AutomationAdaptable on many liquid handlers.

Thermo KingFisherBioSprint (Qiagen)Ambion MagMax 96

Beckman FX, Beckman NX Hamilton Microlab Star

Hamilton StarLET

Xiril Neon InstrumentsCaliper Sciclone

Perkin Elmer Janus

Agilent Bravo



CleanNGS

Unique Features• Optimized buffer system

optimizing size selection• Faster separations due to

new beads• Produced RNase free• Suitable for both DNA as

well as RNA purifications

Kit advantages• Carboxyl beads, providing

highest purity & recovery (yield)

• Simple 3 step process• Designed for automation• No centrifugation / filtration• 1 on 1 replacement for:

– AMPureXP

– RNACleanXP


CleanNGS market opportunities• Opportunities:

– NGS market– Single Cell Sequencing market– Oxford Nanopore users– MicroArray facilities


CleanNGS vs CleanPCR• What is new and better in CleanNGS

– Updated for best performance with latest library prep procedures

– Best size selection efficiency

– Higher magnetite content, providing faster separation times

– RNase free production process allowing for usage in all RNA applications


CleanNGS features• Eliminates primers, salts and nucleotides

• Binds 100bp fragments and larger (typical 100bp-10kb)

• Flexible – can use partial or full plates- Can be performed in PCR microtiter plate if <70ul- Plate transfer to larger capacity plate required if >70ul- 96- & 384-well formats

Kit Components• CleanNGS beads in PCR binding buffer

User-Supplied Components• Ethanol• ring magnet plate purchased from CleanNA• TE or reagent-grade water for DNA elution


CleanNGS – PCR clean-up process

1. Add CleanNGS and Mix to bind NGS library fragments or PCR products to the magnetic beads.

2. Magnetize the beads to separate the sample from the contaminates. Aspirate Solution containing contaminants.

3. Add 70% Ethanol to wash the beads. Magnetize the beads. Aspirate the Ethanol. Repeat step.

4. Add Water to Elute DNA from bead. Magnetize the beads and draw out purified PCR product.

1 2 3 4


CleanNGS - size selection process

1. Add the first volume of CleanNGS to library to bind large fragments2. Separate the beads from solution using a magnet3. Transfer the supernatant containing the smaller fragments to a clean well4. Add the second volume of CleanNGS to bind the size fragments of interest5. Separate the beads from solution using a magnet, aspirate and discard the supernatant with

contaminants6. Wash the beads using 80% ethanol7. Add Water to Elute DNA from bead. Separate the beads using a magnet and draw out purified and

size selected DNA fragments.

1 2 3 4 5 6 7


Double Sided Size Selection• 0.8x/0.7x (left/right) size selection• Sample volume 50 µL

• 1st Step: Add 0.7 x 50 µL = 35 µL CleanNGS• Binding large fragments onto the beads

• Transfer supernatant after binding and separation

• 2nd Step: Add (0.8-0.7) x 50 µL = 5 µL CleanNGS to supernatant• Bind fragment of interest to the new set of beads

• Aspirate and discard the supernatant (contains smallest fragments)• Wash and elute the size selected DNA

Example


Binding Large Fragments• 1st Step: Add 0.7 x 50 µL = 35 µL CleanNGS• Binding large fragments onto the beads

• Transfer supernatant after binding and separation

Fragments of interest

Fragments to bind


Binding Fragments of Interest• 2nd Step: Add (0.8-0.7) x 50 µL = 5 µL CleanNGS to supernatant• Bind fragment of interest to the new set of beads


Fragments to bind


Cleaning Fragments of Interest• Aspirate and discard the supernatant (contains smallest fragments)• Wash and elute the size selected DNA




Double-Sided Size SelectionSheared gDNA was used for a double-sided size selection using 0.8x/0.7x ratio’s (left/right) using CleanNA’s CleanNGS versus company A following the manufacturer’s recommended protocols and eluted in 25 µL. The DNA was analyzed using an Agilent TapeStation 2200.

Average bp selectedCleanNGS 348 bpCompany A 350 bp


Total RNA Purification

A volume of 10 µL of total RNA with 500 and 50 ng/µL was purified using CleanNA’s CleanNGS, following manufacturer’s recommended protocol.

The purified RNA was eluted in 20 µL and analyzed using Agilent’s TapeStation 2200.

Total RNA recovery was 86-94% respectively.


50 bp ladder purification

10 µL of a 50 bp ladder was purified using CleanNA’s CleanNGS versus Company A according the manufacturer’s protocols.

Purified DNA was eluted in 20 µL.

DNA was analyzed using Agilent’s Tapestation 2200.


CleanNGS vs AMPureXP

• All Samples are from the same origin (both sample as well as concentration)

• Concentration + size analysis have been determined using the Tapestation.

– 1.8x bead purification: To accomplish a complete purification of all fragment sizes/lenghts

– 1.0x bead purification:Primers + small amplicons are being removed

– 0.65x – 0.25x bead purification: Size selectionBoth small as well as large fragments are being removed


1.8x Beads – Complete Purification


1.0x Beads – Removal of small Fragments


0.65x – 0.25x Beads – Size Selection


Test setup using Roche Protocol• 8 libraries (WB1 – WB8)• Split after preparation (exact duplicates)• Purified using the Roche protocol

– One set utilized CleanNGS– One set utilized AMPureXP

• i.e. selection of fragments 300bp and larger• Sample check on the BioAnalyzer 2100


Recovery after size selectionCleanNGS Competitor A

WB1 340 pg/µl 231 pg/µl









CleanNGS vs competitor AMethod [% yield] Nanodrop [% yield] Qubit

No purification 100 100Comp Z (colomns) 46 45

CleanNA 73 76Comp A 58 61


Documents

Magnetic Bead Based Nucleic Acid Cleanup...2018/03/13 · nucleic acids • Paramagnetic Beads − Silica beads: use anion exchange principles for nucleic acid isolation − CleanNA