MagnaMedics
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MagSi-NGSPREP Plus
Art.No.
MDKT0001

Product Manual

Version 1.0 | 03/09/2015

MagnaMedics
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MagSi-NGSPREP Plus
Clean-up and size selection in sample preparation for NGS library construction

This product is for R&D use only. Not for drug, household or other uses. For more information, please
consult the appropriate Material Safety Data Sheet (MSDS), available on our website atwww.magnamedics.com

Table of Contents
1. Introduction........................................................................................................................3
1.1 Summary of MagSi-NGSPREP Plus uses and protocols.................................................................................3
1.2 General information.............................................................................................................................4

2. MagSi-NGSPREP Plus contents................................................................................................5
2.1 Materials Supplied by the User..............................................................................................................5

3. Kit Usage............................................................................................................................ 6
3.1 Preparations before use.......................................................................................................................6
3.2 Handling guidelines for MagSi-NGSPREP Plus.............................................................................................6
3.3 Safety information...............................................................................................................................6

4. Protocols.............................................................................................................................7
4.1 Clean-up of enzymatic reactions............................................................................................................7
4.2 Left Side Size Selection........................................................................................................................8
4.3 Right Side Size Selection......................................................................................................................9
4.4 Double Sided Size Selection................................................................................................................11

5. Troubleshooting................................................................................................................132PM0006-100_MagSi-NGS^PREP_Plus

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1. Introduction
1.1 Summary of MagSi-NGSPREP Plus uses and protocols
A. General-purpose DNA clean-up (Protocol 4.1)
For clean-up of DNA fragment (without size selection) following all DNA manipulation procedures*. MagSi-NGS PREP
Plus efficiently captures and purifies all double stranded DNA fragments greater than about 100 bp in size.
This protocol is applicable to:•

PCR amplification procedures•

cDNA constructions•

DNA restriction digestions•

General purpose DNA manipulation, concentration, and buffer exchange procedures

B. DNA Size selection•

Left Side Size Selection (Protocol 4.2)
This protocol removes DNA fragments below a target size. Target DNA is bound to the beads, washed andeluted. This protocol is applicable in various steps of NGS library preparation procedures.

•

Right Side Size Selection (Protocol 4.3)
This protocol removes DNA fragments above a target size. DNA above the target size is bound to thebeads, leaving target DNA in solution. In a second binding step, target DNA is bound to the beads,
washed and eluted.

•

Double Sided Size Selection (Protocol 4.4)
This protocol removes DNA fragments above and below target size range. DNA above the target size isbound to the beads, leaving target DNA in solution. In a second binding step, DNA below target size is leftin solution while target DNA is bound to the beads, washed and eluted.

* MagSi-NGSPREP Plus can replace the use of Agencourt® AMPure® XP in SPRI procedures without affecting
protocols and automation.

¹ Agencourt® and AMPure® XP are registered trademarks by Beckman Coulter, Inc.
² All other trademarks cited here are the property of their respective owners.

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1.2 General information
MagSi-NGSPREP Plus provides a convenient tool for ultra-fast and efficient purification and size selection of
DNA products. DNA fragments will be bound directly onto the surface of the magnetic beads, leaving
unincorporated nucleotides, primers, primer dimers, and other contaminants in solution. Finally, the DNA
fragments are eluted with low salt buffer or reagent grade water.
The technology for binding of DNA fragments onto the applied magnetic nanoparticle surface does not
require use of any hazardous chaotropic buffers. The purification protocols are optimized to provide highyield and purity of the recovered DNA fragments.
MagSi-NGSPREP Plus allows either non-selective binding, or size-targeted binding of double-stranded DNA
fragments ranging from 80 – 1000 bp with specific reagent volume:sample volume ratio's. By increasing
the volume of MagSi-NGSPREP Plus, the efficiency of binding smaller fragments increases. This enables theuser to selectively keep or discard undesired fragment sizes.
Depending on which protocol is used, the total preparation time is 20-30 minutes and the hands-on timenecessary for the whole procedure is reduced to a minimum. The kit can be used manually and on
automated workstations using single tubes, or 96- and 384-well PCR plates.
The kit is stable for 1 year when stored at 2-8°C.

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2. MagSi-NGSPREP Plus contents
Article Number

MDKT00010005

MDKT00010075

MDKT00010500

Kit size

5 mL

75 mL

500 mL

Product Manual

1

1

1

2.1 Materials Supplied by the User
Consumables & Equipment

Multichannel pipettes

PCR plates

20 µL and 200 µL
96-well PCR Plates, (suggested: ABgene, Cat.No.: AB-0800, AB-1000 or AB-1400 )384-well PCR Plates, (suggested: ABgene, Cat.No.: AB-1111)
MM-Separator M96 (MagnaMedics, Art.No.: MD90002): Magnetic separator for 96-well
microplates and PCR plates.
MM-Separator PCR strip adapter (MagnaMedics, Art.No.: MD90003): Adapter for manual
use with 8- and 12-tube PCR strips and is complementary to MM-Separator M96.

Magnetic separator

MM-Separator 96 SBS (MagnaMedics, Art.No.: MD90005): Magnetic separator for 96-well
microplates and PCR plates, suitable for automated processes
MM-Separator 384 SBS (MagnaMedics, Art.No.: MD90006): Magnetic separator for 384-well
microplates and PCR plates. suitable for automated processes

Reagents

Pure Ethanol p.a.

VWR cat# 1.00013.1000

Elution buffer

Molecular biology grade water, TRIS (10 mM, pH 8.0) or TE-buffer

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3. Kit Usage
3.1 Preparations before use•

Prepare an ethanol solution freshly (!):
For Protocol 4.1, prepare a 70% ethanol solution.
For protocol 4.2, 4.3 and 4.4, prepare a 85% ethanol solution.

•

Before use, vortex the MagSi-NGSPREP Plus intensively into a homogeneous suspension.

3.2 Handling guidelines for MagSi-NGSPREP Plus•

Drying time may vary due to differences in the laboratory environment. Optionally increase the drying
time a little until the wells appear to be dry. Caution: Do not over-dry the beads, this can result in loss ofrecovery.

•

When transferring purified samples, preferably leave 2-5 µL liquid behind in order to prevent carryover ofmagnetic beads into the final plate. If beads are present in final sample, perform the separation again.

3.3 Safety information
Wear a suitable lab coat, disposable gloves and protective goggles. For more information, please consult theappropriate material safety data sheets (MSDS). These are available online at www.magnamedics.com.

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4. Protocols
4.1 Clean-up of enzymatic reactions
1.

Before use, vortex MagSi-NGS PREP Plus to fully resuspend the beads.

2.

Add MagSi-NGS PREP Plus according to the table below; mix by pipetting up and down until ahomogeneous suspension is obtained. Incubate for 5 minutes to allow beads to bind the DNA.
Reaction volume (µL)

MagSi-NGS PREP Plus volume (µL)

10

18

15

27

20

36

25

45

50

90
For different reaction volumes, use the following equation:
Volume of MagSi-NGSPREP Plus = 1.8 x Reaction Volume

3.

Place the sample plate on the magnetic separator for 2 minutes to collect the magnetic beads
until a clear solution is obtained.

4.

Remove the cleared supernatant from the beads and discard.
This step must be performed while the plate is on the magnet. Remove the supernatant as much as
possible, but make sure you do not disturb the magnetic beads.

5.

Add 180 µL EtOH 70% and incubate for 30 seconds to allow the beads to settle to the magnet again.
This step can be performed while the plate is placed on the magnet. (It is not necessary to resuspend thebeads).

6.

Discard the supernatant.
This step must be performed while the plate is on the magnet.

7.

Repeat steps 5-6 once more for a total of 2 washing steps.

8.

Air-dry the magnetic particles for approximately 5 minutes.
This step can be performed while the plate is placed on the magnet.

9.

Remove the plate from the magnet and add 40 µL Elution Buffer. Mix by pipetting 10x and incubate for2 minutes to elute.

10.

Place the sample plate on the magnetic separator for 1 minute to collect the magnetic beads .

11.

Transfer the supernatant to the final plate.
This step must be performed while the plate is on the magnet. Leave 5 µL liquid behind to prevent
transfer of beads into the final plate.

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4.2 Left Side Size Selection
Decreasing the ratio of MagSi-NGSPREP Plus volume to sample volume for Left Side Selection will decrease theefficiency of binding smaller fragments (see Figure 1).

Figure 1: Left Side Size Selection with given ratios of Magsi-NGSPREP Plus volume to sample volume
Volume of sample * Ratio = Volume of MagSi-NGSPREP Plus
Example: 50 µL * 0.65 = 32.5 µL of MagSi-NGSPREP Plus

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1.

Before use, vortex MagSi-NGS PREP Plus to fully resuspend the beads.

2.

Add the required volume of MagSi-NGSPREP Plus for the desired ratio to the sample. Mix by pipetting 10x
until a homogeneous suspension is obtained and incubate for 5 minutes.

3.

Place the sample plate on the magnetic separator for 2 minutes to collect the magnetic beads
until a clear solution is obtained.

4.

Remove the cleared supernatant from the beads and discard.
This step must be performed while the plate is on the magnet. Remove the supernatant as much as
possible, but make sure you do not disturb the magnetic beads.

5.

Add 180 µL EtOH 85% and incubate for 30 seconds to allow the beads to settle to the magnet again.
This step can be performed while the plate is placed on the magnet.

6.

Discard the supernatant.
This step must be performed while the plate is on the magnet.

7.

Repeat steps 5-6 once more for a total of 2 washing steps.

8.

Air-dry the magnetic particles for approximately 5 minutes.
This step can be performed while the plate is placed on the magnet.

9.

Remove the plate from the magnet and add 40 µL Elution Buffer. Mix by pipetting 10x and incubate for2 minutes to elute.

10.

Place the sample plate on the magnetic separator for 1 minute to collect the magnetic beads .

11.

Transfer the eluate (size selected sample) to the final plate.
This step must be performed while the plate is on the magnet. Leave 2-5 µL liquid behind to prevent
transfer of beads into the final plate.

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4.3 Right Side Size Selection
Increasing the ratio of MagSi-NGSPREP Plus volume to sample volume for Right Side Selection will increase theefficiency of removing larger fragments (see Figure 2).

Figure 2: Right Side Size Selection with given ratios of MagSi-NGS PREP Plus volume to sample volume
Volume of sample * Ratio = Volume of MagSi-NGSPREP Plus
Example: 50 µL * 0.65 = 32.5 µL of MagSi-NGSPREP Plus

1.

Before use, vortex MagSi-NGS PREP Plus to fully resuspend the beads.

2.

Add the required volume of MagSi-NGSPREP Plus for the desired ratio to the sample. Mix by pipetting 10x
until a homogeneous suspension is obtained and incubate for 5 minutes.

3.

Place the sample plate on the magnetic separator for 2 minutes to collect the magnetic beads
until a clear solution is obtained.

4.

Transfer the cleared supernatant from the beads to a new container.
Be careful not to aspirate beads, as these contain the undesired larger fragment sizes.

5.

Add the required volume of MagSi-NGSPREP Plus using the following formula:
Initial sample volume * (1.8x – Right Side ratio) = Volume of MagSi-NGSPREP Plus

Mix by pipetting 10x until a homogeneous suspension is obtained and incubate for 5 minutes.
6.

Place the sample plate on the magnetic separator to collect the magnetic beads until a clear solution is
obtained. Collection times may vary; a larger initial sample volume, bigger MagSi-NGSPREP ratio or
weaker magnet will result in longer collection times.

7.

Remove the cleared supernatant from the beads and discard.
This step must be performed while the plate is on the magnet. Remove the supernatant as much as
possible, but make sure you do not disturb the magnetic beads.

8.

Add 180 µL EtOH 85% and incubate for 30 seconds to allow the beads to settle to the magnet again.
This step can be performed while the plate is placed on the magnet.

9.

Discard the supernatant.
This step must be performed while the plate is on the magnet.

10.

Repeat steps 8-9 once more for a total of 2 washing steps.

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11.

Air-dry the magnetic particles for approximately 5 minutes.
This step can be performed while the plate is placed on the magnet.

12.

Remove the plate from the magnet and add 40 µL Elution Buffer. Mix by pipetting 10x and incubate for2 minutes to elute.

13.

Place the sample plate on the magnetic separator for 1 minute to collect the magnetic beads .

14.

Transfer the eluate (size selected sample) to the final plate.
This step must be performed while the plate is on the magnet. Leave 2-5 µL liquid behind to prevent
transfer of beads into the final plate.

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4.4 Double Sided Size Selection
The Left Side Selection ratio is always greater than the Right Side Selection ratio. In the first step (Right Side
Selection), increasing the ratio of MagSi-NGSPREP Plus volume to sample volume will increase the efficiency ofremoving larger fragments. In the second step (Left Side Selection), decreasing the ratio of MagSi-NGS PREP Plus
volume to sample volume will increase the efficiency of removing smaller fragments (see Figure 3).

Figure 3: Double Sided Size Selection with given ratio's of MagSi-NGSPREP Plus volume to sample volume
Volume of sample * Ratio = Volume of MagSi-NGSPREP Plus
Example: 50 µL * 0.65 = 32.5 µL of MagSi-NGSPREP Plus

1.

Before use, vortex MagSi-NGS PREP Plus to fully resuspend the beads.

2.

Add the required volume of MagSi-NGSPREP Plus for the desired Right Side Ratio to the sample. Mix bypipetting 10x until a homogeneous suspension is obtained and incubate for 5 minutes.

3.

Place the sample plate on the magnetic separator for 2 minutes to collect the magnetic beads
until a clear solution is obtained.

4.

Transfer the cleared supernatant from the beads to a new container.
Be careful not to aspirate beads, as these contain the undesired larger fragment sizes.

5.

Add the required volume of MagSi-NGSPREP Plus using the following formula:
Initial sample volume * (Left Side ratio – Right Side ratio) = Volume of MagSi-NGS PREP Plus

Mix by pipetting 10x until a homogeneous suspension is obtained and incubate for 5 minutes.
6.

Place the sample plate on the magnetic separator to collect the magnetic beads until a clear solution is
obtained. Collection times may vary; a higher initial sample volume, higher MagSi-NGS PREP Plus ratio or
weaker magnet will result in longer collection times.

7.

Remove the cleared supernatant from the beads and discard.
This step must be performed while the plate is on the magnet. Remove the supernatant as much as
possible, but make sure you do not disturb the magnetic beads.

8.

Add 180 µL EtOH 85% and incubate for 30 seconds to allow the beads to settle to the magnet again.
This step can be performed while the plate is placed on the magnet.

9.

Discard the supernatant.
This step must be performed while the plate is on the magnet.

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10.

Repeat steps 8-9 once more for a total of 2 washing steps.

11.

Air-dry the magnetic particles for approximately 5 minutes.
This step can be performed while the plate is placed on the magnet.

12.

Remove the plate from the magnet and add 40 µL Elution Buffer. Mix by pipetting 10x and incubate for2 minutes to elute.

13.

Place the sample plate on the magnetic separator for 1 minute to collect the magnetic beads .

14.

Transfer the eluate (size selected sample) to the final plate.
This step must be performed while the plate is on the magnet. Leave 2-5 µL liquid behind to prevent
transfer of beads into the final plate.

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5. Troubleshooting
Troubleshooting guidelines for MagSi-NGSPREP Plus
ProblemPossible cause
Insufficient binding of DNA

Overdrying of beads

Suggestion- Increase pipette mixing steps- Increase binding incubation time

- Decrease drying time to a minimum for removal of
traces of ethanol. Visually inspect for leftover liquid.

Low recovery of DNA
Insufficient EtOH removal after
washing steps

Insufficient elution

Insufficient removal of

Unwanted products in wells or on

unwanted reaction products

beads

Magnetic beads in final eluate

Carryover of beads into finalcontainer- Make sure to discard all wash solution- Increase drying time

- Increase pipette mixing steps- Increase elution incubation time

- For washing, use the maximum working volume- Increase pipette mixing steps for washing- Leave 5 µL of liquid behind to prevent beads frombeing aspirated- Decrease aspiration speed of pipetting- After dispensing onto sample, immediately mix toInsufficient mixing of sample and
MagSi-NGSPREP Plusprevent differences in buffer concentration within thesample- Increase mix steps after addition of MagSi-NGSPREP
Plus

Insufficient binding efficiency inUndesired fragment sizes

removal of undesired fragments- Increase incubation time for binding of DNA fragments

remaining after Size Selection
Carryover of beads with undesired- Increase incubation time for magnetic separation

fragments- Repeat magnetic separation and transferof samples

Reagents in the sample that effectsize cut-off (e.g. polyethylene
glycol, MgCl2)

PM0006-100_MagSi-NGS^PREP_Plus- Adjust Ratio for size selection- Remove reagents from sample with Protocol 4.1, thenproceed with size selection

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MagnaMedics Diagnostics B.V.
IVD, Food-Diagnostics, Life-Science Products,
Laboratory & Production

De Asselen Kuil 20a
6161 RD Geleen (The Netherlands)
Tel: +31-(0)46-820 0206
Fax: +31-(0)46-410 6825
E-mail: info@magnamedics.com

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1.

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2.

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3.

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4.

End user name and telephone number (if different)

5.

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6.

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7.

Quantity and size of products

8.

Indicate if your account is VAT exempt

There is no minimum order requirement.

Order via e-mail:sales@magnamedics.com

Ordering via fax or telephone:Tel: +31-(0)46-820 0206
Fax: +31-(0)46-410 6825

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