BcMag™ One-Step DNA & RNA Cleanup Kit provides one-step removal of impurity by negative chromatography from pre-purified samples of DNA/RNA. The impurity includes DNA/RNA polymerases, modifying enzymes, restriction endonucleases, ligases, kinases, nucleases, phosphatases, protein, most of the detergent, most of the fluorescent or no fluorescent dyes, divalent cations such as Ca2+, Mg2+, excess primer, dimer, adapter, DNA/RNA fragments (<100- mer ssDNA), free dNTPs/NTPs and as well as and their analogs including radiolabeled, biotinylated and fluorescent derivatives.

The protocol is straightforward: one tube, one step, and one minute. Added magnetic beads directly to the pre-purified DNA/RNA samples and vortex or pipette to capture and remove the impurities. After vortexing/pipetting, the beads are captured by a magnetic Rack, while the supernatant contains the purified and ready-to-run products. The beads are suitable for DNA/RNA fragments, plasmid DNA and genomic DNA.

Features and Advantages

●

Simple protocol: No liquid transfer, One-tube, One-step

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Ultrafast: One-minute protocol

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Higher purity and recovery > 90% DNA

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Effective Cleanup: Removes excess primer (<100- mer ssDNA), dimer, adapter, a salt such as Mg2+, detergent, dNTPs, enzymes, and dye

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Cost-effective: Eliminates columns, filters, laborious repeat pipetting, and ethanol

●

High throughput: Compatible with many different automated liquid handling systems

PROTOCOL

A. Materials Required by the User

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18.2 MΩ.cm, DNase/RNase-Free Ultrapure Water

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Triton™ X-100, Sigma, Catalog No. T8787

●

Others

Item

Magnetic Rack for centrifuge tube

** Based on sample volume, the user can choose one of the following magnetic Racks

Source

•  BcMag™ Rack-2 for holding two individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-01)

•  BcMag™ Rack-6 for holding six individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-02)

•  BcMag™ Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Bioclone, Cat. No. MS-03)

•  BcMag™ Rack-50 for holding one 50 ml centrifuge tube, one 15 ml centrifuge tube, and four individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-04)

Item

BcMag™ 96-well Plate Magnetic Rack.

Source

•  BcMa™ 96-well Plate Magnetic Rack (side-pull) compatible with 96-well PCR plate and 96-well microplate or other compatible Racks (Bioclone, Cat. No. MS-05)

Item

Adjustable Single and Multichannel Pipettes

Item

Centrifuge with Swinging Bucket

Addition items are required if using 96-well PCR plates / tubes

Vortex Mixer

** The user can also use other compatible vortex mixers. However, the Time and speed should be optimized, and the mixer should be: Orbit ≥1.5 mm-4 mm, Speed ≥ 2000 rpm

Eppendorf™ MixMate™

Eppendorf, Cat. No. 5353000529

Tube Holder PCR 96

Eppendorf, Cat. No. 022674005

Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL

Eppendorf, Cat. No. 022674048

Smart Mixer, Multi Shaker

BenchTop Lab Systems, Cat. No. 5353000529

1.5/2.0 mL centrifuge tube

96-well PCR Plates or 8-Strip PCR Tubes

PCR plates/tubes

** IMPORTANT! If using other tubes or PCR plates, make sure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates must be ≥2.5mm.

Items

Magnetic Rack for centrifuge tube

** Based on sample volume, the user can choose one of the following magnetic Racks

Source

●

BcMag™ Rack-2 for holding two individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-01)

●

BcMag™ Rack-6 for holding six individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-02)

●

BcMag™ Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Bioclone, Cat. No. MS-03)

●

BcMag™ Rack-50 for holding one 50 ml centrifuge tube, one 15 ml centrifuge tube, and four individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-04)

BcMag™ 96-well Plate Magnetic Rack

●

BcMa™ 96-well Plate Magnetic Rack (side-pull) compatible with 96-well PCR plate and 96-well microplate or other compatible Racks (Bioclone, Cat. No. MS-05)

Adjustable Single and Multichannel Pipettes

Centrifuge with Swinging Bucket

Addition items are required if using 96-well PCR plates/tubes

Vortex Mixer

** The user can also use other compatible vortex mixers. However, the Time and Speed should be optimized, and the mixer should be: Orbit ≥1.5 mm-4 mm, Speed ≥ 2000 rpm

Eppendorf™ MixMate™

Tube Holder PCR 96

Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL

Smart Mixer, Multi Shaker

Eppendorf, Cat. No. 5353000529

Eppendorf, Cat. No. 022674005

Eppendorf, Cat. No. 022674048

BenchTop Lab Systems, Cat. No. 5353000529

Eppendorf™ MixMate™

Tube Holder PCR 96

Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL

Smart Mixer, Multi Shaker
 

Eppendorf, Cat. No. 5353000529

Eppendorf, Cat. No. 022674005

Eppendorf, Cat. No. 022674048
 

BenchTop Lab Systems, Cat. No. 5353000529

1.5/2.0 mL centrifuge tube

96-well PCR Plates or 8-Strip PCR Tubes

PCR plates/tubes

! IMPORTANT ! If using other tubes or PCR plates, make sure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates must be ≥2.5mm.

B. Procedure

! Important !

●

The following protocol is optimized for the efficient cleanup of 10µl DNA sample. The procedure may need to be optimized if an alternative reaction scale is used.

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Shake or vortex the bottle to completely resuspend the magnetic beads before using.

●

Do not allow the magnetic beads to sit for more than two minutes before dispensing.

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Based on applications, the user should choose buffer conditions based on table 1. For example, if the sample does not contain detergent, add 1 μL of 1% Triton™ X-100 solution to a 10 μL sample (final concentration is 0.1%).

●

Quantification of the nucleic acids: Use only fluorescence methods such as qPCR, Qubit, and Pico Green. OD260 methods such Nanodrop and UV-spectrophotometry are not-suitable.

Table 1 – DNA Fragment Removal

dsDNA (100 bp)[Buffer]

+ 0.1% Triton x-100, pH7.5
No Removal

- 0.1% Triton x-100, pH7.5
Removal

+ 0.1% Triton x-100, pH 8.0
Removal

- 0.1% Triton x-100, pH 8.0
Removal

+ 0.1% Triton x-100, pH 8.8
No Removal

- 0.1% Triton x-100, pH 8.8
RemovaldsDNA (150 bp)[Buffer]

+ 0.1% Triton x-100, pH7.5
No Removal

- 0.1% Triton x-100, pH7.5
Removal

+ 0.1% Triton x-100, pH 8.0
No Removal

- 0.1% Triton x-100, pH 8.0
Removal

+ 0.1% Triton x-100, pH 8.8
No Removal

- 0.1% Triton x-100, pH 8.8
RemovaldsDNA (200 bp)[Buffer]

+ 0.1% Triton x-100, pH7.5
No Removal

- 0.1% Triton x-100, pH7.5
Removal

+ 0.1% Triton x-100, pH 8.0
No Removal

- 0.1% Triton x-100, pH 8.0
Removal

+ 0.1% Triton x-100, pH 8.8
No Removal

- 0.1% Triton x-100, pH 8.8
RemovaldsDNA (300 bp)[Buffer]

+ 0.1% Triton x-100, pH7.5
No Removal

- 0.1% Triton x-100, pH7.5
No Removal

+ 0.1% Triton x-100, pH 8.0
No Removal

- 0.1% Triton x-100, pH 8.0
No Removal

+ 0.1% Triton x-100, pH 8.8
No Removal

- 0.1% Triton x-100, pH 8.8
No RemovalssDNA 100 mer[Buffer]

+ 0.1% Triton x-100, pH7.5
Removal

- 0.1% Triton x-100, pH7.5
Removal

+ 0.1% Triton x-100, pH 8.0
Removal

- 0.1% Triton x-100, pH 8.0
Removal

+ 0.1% Triton x-100, pH 8.8
Removal

- 0.1% Triton x-100, pH 8.8
RemovalPrevious slideNext slide

Table 1 – DNA Fragment Removal

DNA

Buffer

+ 0.1%
Triton x-100
pH7.5

– 0.1%
Triton x-100
pH7.5

+ 0.1%
Triton x-100
pH 8.0

– 0.1%
Triton x-100
pH 8.0

+ 0.1%
Triton x-100
pH 8.8

– 0.1%
Triton x-100
pH 8.8

dsDNA
(100 bp)

No Removal

Removal

Removal

Removal

No Removal

Removal

dsDNA
(150 bp)

No Removal

Removal

No Removal

Removal

No Removal

Removal

dsDNA
(200 bp)

No Removal

Removal

No Removal

Removal

No Removal

Removal

dsDNA
(300 bp)

No Removal

No Removal

No Removal

No Removal

No Removal

No Removal

ssDNA
100 mer

Removal

Removal

Removal

Removal

Removal

Removal

Please Note:

dsDNA – Double-Stranded DNA; ssDNA – Single-stranded DNA

The assay was done by using the following conditions:

1. 10 mM Tris-HCl with or without 0.1% triton (final concentration) and three different: pH 7.5, pH 8.0 and pH 8.8

1.

Add 5 μL magnetic beads to the 10 μL DNA sample.

2.

If necessary, briefly centrifuge at 2500 rpm for 30 seconds to bring all contents to the bottom of the tube.

3.

Mix thoroughly for 1 minute by slowly pipetting up and down 25 times (one minute) or by vortex mixer for 5 minutes at 2500 rpm.

4.

If necessary, briefly centrifuge at 2500 rpm for 30 seconds to bring all contents to the bottom of the tube.

5.

Place the sample plate on the magnetic separation plate for 30 seconds or until the solution is clear to separate beads from the solution.

6.

Transfer the supernatant to a clean plate while the sample plate remains on the magnetic separation plate for downstream applications.

C. Troubleshooting

Problem

Low DNA Recovery

Probable Cause

Vertexing speed is too fast.

Vertexing time is too long.

Suggestion

• Reducing either the speed or time

• If using other digital vortex mixers, the vortex condition, such as speed and time, has to be optimized.

Problem

Low DNA Recovery

Probable Cause

Using too many magnetic beads

Suggestion

Thoroughly resuspend the magnetic beads and use the correct amounts of the beads.

Problem

Failure to Remove Impurities.

Probable Cause

Used inappropriate PCR tubes or PCR plates

Suggestion

Make sure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates is ≥2.5mm.

Problem

Failure to Remove Impurities.

Probable Cause

Vortex speed is too slow, or vortex time is too short.

Suggestion

• Increasing either the speed or time

• If using other digital vortex mixers, the vortex condition, such as speed and time, has to be optimized.

Problem

Failure to Remove Impurities.

Probable Cause

Using fewer magnetic beads

Suggestion

Thoroughly resuspend the magnetic beads and use the correct amounts of the beads.

Problem

Failure to Remove Impurities.

Probable Cause

Strong secondary structure of DNA fragments ( < 50bp dsDNA or < 100 mer ssDNA)

Suggestion

Denature the sample by heating it at 95°C for 2 min.

Problem

Failure to Remove Impurities.

Probable Cause

Too much primer, dimer, adaptor, free dye, and detergent

Suggestion

• Use more magnetic beads.

• Perform the second round of purification by following the same protocol.

Problem

Probable Cause

Suggestion

Low DNA Recovery

Vertexing speed is too fast.

Vertexing time is too long.

• Reducing either the speed or time

• If using other digital vortex mixers, the vortex condition, such as speed and time, has to be optimized.

Using too many magnetic beads

Thoroughly resuspend the magnetic beads and use the correct amounts of the beads.

Failure to Remove Impurities.

Used inappropriate PCR tubes or PCR plates

Make sure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates is ≥2.5mm.

Vortex speed is too slow, or vortex time is too short.

• Increasing either the speed or time

• If using other digital vortex mixers, the vortex condition, such as speed and time, has to be optimized.

Using fewer magnetic beads

Thoroughly resuspend the magnetic beads and use the correct amounts of the beads.

Strong secondary structure of DNA fragments ( < 50bp dsDNA or < 100 mer ssDNA )

Denature the sample by heating it at 95°C for 2 min.

Too much primer, dimer, adaptor, free dye, and detergent

• Use more magnetic beads.

• Perform the second round of purification by following the same protocol.

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