MAGBIO

“WE MAKE NGS BETTER”

HighPrep PCR PB Cleanup and Size Selection

for Long Read Sequencing

Catalog Nos. PB-60001, PB-60005, PB-60050

Manual Revision 0
WI-72-125

Magnetic bead-based chemistry

No centrifugation or filtration
Efficient cleanup and size selection

Protocol
Contents
Product Description ................................................................................... 1
Protocol: Amplicon Cleanup for SMRTbell Library Preparation .. 2-3
Protocol: DNA Purification ....................................................................... 4-5
Protocol: DNA Size Selection ................................................................... 6-7
Ordering and ReIated Product Information ....................................... 8

For research use only

Information in this document is subject to change without notice.

MAGBIO GENOMICS, INC. DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A
PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL MAGBIO GENOMICS, INC. BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON
ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO
THE USE THEREOF,WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT MAGBIO GENOMICS, INC. IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES.

TRADEMARKS
The trademarks mentioned herein are the property of MagBio Genomics, Inc. or their respective owners.
 HighPrep PCR PB

Product Description
HighPrep PCR PB is a paramagnetic bead-based reagent used in amplicon cleanup, DNA concentration,
and purification. It can also be diluted with low salt buffer (Elution Buffer) to 35% (v/v) and then used
for size selection of SMRTbell libraries.

Preparation
• DO NOT FREEZE
• Keep at room temperature for 30 min. prior to use
• Thoroughly shake the HighPrep PCR PB to resuspend the beads before use
• HighPrep PCR PB diluted with Elution Buffer to 35% (v/v) is stable for 2 months when stored at 2-8°C

Materials Supplied in the Kit

• HighPrep PCR PB

Kit Contents
HighPrep PCR PB PB-60001
(Sample)
PB-60005 PB-60050 Storage
Catalog No.
HighPrep PCR PB1
1 mL 5 mL 50 mL 2-8°C
volume
Elution Buffer Must be purchased separately. See ordering and related
2 mL 15-25°C
volume product information on page 8.

Shipping and Storage

1
• HighPrep PCR PB ships at room temperature. Store at 2-8°C.

Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate safety data sheets (SDSs). The SDS can
be downloaded from the “Product Documents” tab when viewing this product at
www.magbiogenomics.com.

US/Canada: +1 301-302-0144 | Web: www.magbiogenomics.com | E-mail: [email protected]

Europe: +49 7250 33 13 403 | Web: www.magbiogenomics.com | E-mail: [email protected]

1
 HighPrep PCR PB

Protocol: Amplicon Cleanup for SMRTbell Library Preparation

HighPrep PCR PB is based on paramagnetic bead technology designed for efficient purification
of amplicons prior to SMRTbell library preparation. The purification of amplicon template consists
of removal of salts, PCR buffers, excess primers, primer-dimers, and dNTPs. DNA fragments are
selectively bound to the magnetic bead particles, and highly purified amplicons are eluted with
Tris-HCl buffer (10 mM Tris-HCl, pH 8.5 – pH 9.0) which can be used in library preparation.

Equipment and Reagents to Be Supplied by the User

80% Ethanol (Prepared from non denatured Ethanol)
70% Ethanol (Prepared from non denatured Ethanol)
Elution Buffer (10 mM Tris-HCl pH 8.5-9.0) or MagBio Genomics’ Elution Buffer (see page 8)
Magnetic separation device compatible with 96 or 384 well PCR plate (see page 8)
Magnetic separation device compatible with 1.5 mL/1.7 mL/2 mL microcentrifuge tubes (see page 8)
96-well PCR plate or 384-well PCR plate
1.5 mL LoBind microcentrifuge tubes
Wide-bore pipette tips

Things to do Before Starting

Use the table below to determine the HighPrep PCR PB bead ratio which is required to select
the desired insert size range
Make sure the HighPrep PCR PB is equilibrated at room temperature before use

Insert Size Range (bp) HighPrep PCR PB Bead Ratio/Volume Needed Per Sample

100-300 1.8X*
300-750 1.0X
750-3,000 0.6X
3,000-10,000
0.45X
15,000
* The X represents the volume of the PCR sample to be purified. The required volume of HighPrep PCR PB is calculated as follows:
for a 100-300 bp insert size, required volume of HighPrep PCR PB = 1.8 X (PCR reaction volume)

Protocol
1. Mix the HighPrep PCR PB and amplicon based on the bead ratio in the above table. Mix thoroughly
10-15 times using wide-bore pipette tips.

Tip: Pipette a minimum of 15 μL of beads in order to get the best results. When calculating the
amount of beads to use, if the volume is less than 15 μL then increase the sample volume to reach a
bead volume above 15 μL.

2. Incubate the sample at room temperature for 15 min.

3. Briefly spin down the sample to collect the beads that may have splashed to the sides.

4. Put the tube/plate on a magnetic separation device to magnetize the beads. Wait for the solution to
become clear.

US/Canada: +1 301-302-0144 | Web: www.magbiogenomics.com | E-mail: [email protected]

Europe: +49 7250 33 13 403 | Web: www.magbiogenomics.com | E-mail: [email protected]

2
 HighPrep PCR PB

5. With the sample tube/plate still on the magnetic separation device, remove and transfer the
supernatant to another tube/plate. Avoid bead disturbance while pipetting out the supernatant
(save the supernatant in case you may need this sample).

6. With the sample tube/plate on the magnetic separation device, add 200 μL of 80% Ethanol to each
tube/well, and incubate for 30 seconds at room temperature.
! Make sure to use freshly prepared 80% Ethanol to achieve the best results.

7. With the tube/plate still on the magnetic separation device, remove and discard the supernatant by
pipetting.

8. Repeat steps 6 and 7 for a total of two 80% Ethanol washes.

9. Ensure that there is no remaining 80% Ethanol in the sample tube/well. If droplets are present, do the
following:

i. Spin the sample tube or plate down to bring the droplets down
ii. Place the sample on the magnetic separation device
iii. Use a fine pipette tip to remove the droplets without touching the beads

10. Air dry the beads for 30 to 60 seconds. Remove the sample plate/tube from the magnetic separation
device and add 40 μL of elution buffer, 10 mM Tris-HCl pH 8.5-9.0, or MagBio Genomics’ Elution Buffer
(see page 8).

11. Vortex for 1-2 min. at 2,000 rpm. Spin down the sample tube/plate and place it on the magnetic
separation device.

12. Wait for the solution to be clear and pipette out the supernatant to a new 1.5 mL Lo-Bind tube.

13. Discard the beads.

14. Store the DNA at -20°C or proceed to the next step of library preparation.

US/Canada: +1 301-302-0144 | Web: www.magbiogenomics.com | E-mail: [email protected]

Europe: +49 7250 33 13 403 | Web: www.magbiogenomics.com | E-mail: [email protected]

3
 HighPrep PCR PB

Protocol: DNA Purification

Improvement of DNA Purity (A260/A230)
The quality of DNA can be a reflection on sequencing results. High quality and purity of DNA is of
utmost importance in long read sequencing. HighPrep PCR PB can be used to cleanup genomic
DNA of low sample purity (low A260/A230). Low A260/A230 of DNA can be attributed to carryover
contaminants like salts, carbohydrates etc. The A260/A230 value should be between 2.0 and 2.2
to obtain higher quality libraries. Use HighPrep PCR PB at a ratio of 0.45X for DNA purification to
improve purity,
Purification of Samples Treated with RNase A
If RNA is present in a DNA sample, RNase A treatment is needed prior to library preparation.
After RNase A treatment, use HighPrep PCR PB at a 0.45X ratio for DNA purification.

Concentration of a Dilute DNA Sample Using HighPrep PCR PB

If the sheared DNA that is going to be used for SMRTbell library preparation is dilute, less than
the required concentration of 140 ng/μL, then sheared genomic DNA can be concentrated using
HighPrep PCR PB. Use the 0.45X ratio for concentration of a dilute DNA sample using HighPrep
PCR PB.

Equipment and Reagents to Be Supplied by the User

80% Ethanol (Prepared from non denatured Ethanol)
70% Ethanol (Prepared from non denatured Ethanol)
Elution Buffer (10 mM Tris-HCl pH 8.5-9.0) or MagBio Genomics’ Elution Buffer (see page 8)
Magnetic separation device compatible with 96 or 384 well PCR plate (see page 8)
Magnetic separation device compatible with 1.5 mL/1.7 mL/2 mL microcentrifuge tubes (see page 8)
96-well PCR plate or 384-well PCR plate
1.5 mL LoBind microcentrifuge tubes
Wide-bore pipette tips
Optional: RNase A (if RNA is present in DNA samples)

Things to do Before Starting

Make sure the HighPrep PCR PB is equilibrated at room temperature before use

Protocol
1. Mix the HighPrep PCR PB and DNA solution based on a bead ratio of 0.45X. Mix thoroughly 10-
15 times using wide-bore pipette tips.

2. Incubate the sample at room temperature for 15 min.

3. Briefly spin down the sample to collect the beads that may have splashed to the sides.

4. Put the tube/plate on a magnetic separation device to magnetize the beads. Wait for the solution
to become clear.

5. With the sample tube/plate still on the magnetic separation device, remove and transfer the
supernatant to another tube/plate. Avoid bead disturbance while pipetting out the supernatant
(save the supernatant in case you may need this sample).

US/Canada: +1 301-302-0144 | Web: www.magbiogenomics.com | E-mail: [email protected]

Europe: +49 7250 33 13 403 | Web: www.magbiogenomics.com | E-mail: [email protected]

4
 HighPrep PCR PB

6. With the sample tube/plate on the magnetic separation device, add 200 μL of 70% Ethanol to each
well/tube, and incubate for 30 seconds at room temperature.
! Make sure to use freshly prepared 70% Ethanol to achieve the best results.

7. With the tube/plate still on the magnetic separation device, remove and discard the supernatant by
pipetting.

8. Repeat steps 6 and 7 for a total of two 70% Ethanol washes..

9. Ensure that there is no remaining 70% Ethanol in the sample tube/well. If droplets are present, do
the following:

I. Spin the sample tube or plate down to bring the droplets down
II. Place the sample on the magnetic separation device
III. Use a fine pipette tip to remove the droplets without touching the beads

10. Air dry the beads for 30 to 60 seconds. Remove the sample tube/plate from the magnetic separation
device and add 40 μL of elution buffer, 10 mM Tris-HCl pH 8.5-9.0, or MagBio Genomics’ Elution
Buffer (see page 8).

11. Vortex for 1-2 min. at 2,000 rpm. Spin down the sample tube/plate and place it on the magnetic
separation device.

12. Wait for the solution to be clear and pipette out the supernatant to a new 1.5 mL Lo-Bind tube.

13. Discard the beads.

14. Store the DNA at -20°C or proceed to the next step of library preparation.

US/Canada: +1 301-302-0144 | Web: www.magbiogenomics.com | E-mail: [email protected]

Europe: +49 7250 33 13 403 | Web: www.magbiogenomics.com | E-mail: [email protected]

5
 HighPrep PCR PB

Protocol: DNA Size Selection

Size selection is most effective when the DNA sample or SMRTbell libraries to be size selected are
at a lower concentration of 0.5-10 ng/μL. It is important to adjust sample concentration before
size selection to have efficient size selection. Using highly concentrated DNA outside of the
range given will result in the beads binding to small fragments that are undesirable in long read
sequencing. HighPrep PCR PB is diluted first to a concentration of 35% (v/v) using Elution Buffer.

Dilution of HighPrep PCR PB with Elution Buffer to 35% (v/v)

The final HighPrep PCR PB bead concentration is vital to the success of this procedure. Therefore,
accurate pipetting is necessary in order to achieve a final 35% (v/v) HighPrep PCR PB bead solution.

Before performing size selection, determine the number of reactions needed and the volume of
beads and Elution Buffer needed to dilute HighPrep PCR PB to 35% (v/v).
Reagent Volume (mL)
HighPrep PCR PB 3.5
Elution Buffer 6.5
Total Volume 10

Ratio of HighPrep PCR PB 35% (v/v) Used in DNA Size Selection

SMRTbell Template Length Removal HighPrep PCR PB 35% (v/v) Volume Needed
Remove < 3kb 3.7X
Remove < 4kb 3.3X
Remove < 5kb 3.1X
* The X represents the volume of the sample to be selected. The required volume of HighPrep PCR PB is calculated as follows:
for removal of < 3kb, the required volume of HighPrep PCR PB = 1.8 X (sample volume)

Equipment and Reagents to Be Supplied by the User

80% Ethanol (Prepared from non denatured Ethanol)
70% Ethanol (Prepared from non denatured Ethanol)
Elution Buffer (10 mM Tris-HCl pH 8.5-9.0) or MagBio Genomics’ Elution Buffer (see page 8)
Magnetic separation device compatible with 1.5 mL/1.7 mL/2 mL microcentrifuge tubes (see page 8)
1.5 mL LoBind microcentrifuge tubes
Wide-bore pipette tips
1X dsDNA HS Assay kit
Qubit 2.0, 3.0, or 4.0 fluorometer

Things to do Before Starting

Dilute HighPrep PCR PB with Elution Buffer to 35% (v/v)
Make sure the HighPrep PCR PB is equilibrated at room temperature before use

US/Canada: +1 301-302-0144 | Web: www.magbiogenomics.com | E-mail: [email protected]

Europe: +49 7250 33 13 403 | Web: www.magbiogenomics.com | E-mail: [email protected]

6
 HighPrep PCR PB

Protocol
1. Mix the HighPrep PCR PB and DNA solution based on the ratios shown in the table on page 6
(Ratio of HighPrep PCR PB 35% (v/v) used in DNA size selection). Mix thoroughly 10-15 times using
wide-bore pipette tips.

2. Incubate the sample at room temperature for 15 min.

3. Briefly spin down the sample to collect the beads that may have splashed to the sides.

4. Put the tube on a magnetic separation device to magnetize the beads. Wait for the solution to
become clear.

5. With the sample tube still on the magnetic separation device, remove and transfer the supernatant
to another tube. Avoid bead disturbance while pipetting out the supernatant (save the supernatant
in case you may need this sample).

6. With the sample tube on the magnetic separation device, add 200 μL of 80% Ethanol to each tube
and incubate for 30 seconds at room temperature.
! Make sure to use freshly prepared 80% Ethanol to achieve the best results.

7. With the tube still on the magnetic separation device, remove and discard the supernatant by
pipetting.

8. Repeat steps 6 and 7 for a total of two 80% Ethanol washes.

9. Ensure that there is no remaining 80% Ethanol in the sample tube. If droplets are present, do the
following:

I. Spin the sample tube down to bring the droplets down

II. Place the sample on the magnetic separation device
III. Use a fine pipette tip to remove the droplets without touching the beads

10. Remove the sample tube from the magnetic separation device. Add 10 μL of elution buffer,
10 mM Tris-HCl pH 8.5-9.0, or MagBio Genomics’ Elution Buffer (see page 8).

11. Perform warm elution by heating the sample tube at 37°C for 15 min. to elute the DNA from the
beads.

12. Briefly spin down the sample and place the sample tube on the magnetic separation device. Wait for
the solution to be clear and pipette out the supernatant to a new 1.5 mL Lo-Bind tube.

13. Discard the beads.

14. Measure SMRTbell library concentration using 1 μL of the 10 μL eluted DNA. Use the Qubit
fluorometer and the dsDNA HS Assay Kit according to the manufacturer’s instructions.

15. Store the remaining 9 μL SMRTbell library at -20°C.

US/Canada: +1 301-302-0144 | Web: www.magbiogenomics.com | E-mail: [email protected]

Europe: +49 7250 33 13 403 | Web: www.magbiogenomics.com | E-mail: [email protected]

7
 HighPrep PCR PB

Ordering
HighPrep PCR PB
Catalog No. Product

PB-60005 HighPrep PCR PB (5 mL)

PB-60050 HighPrep PCR PB (50 mL)

Related Products
Elution Buffer
Catalog No. Product

EB-20 Elution Buffer (20 mL)

EB-250 Elution Buffer (250 mL)

High Molecular Weight DNA Isolation Kit

Catalog No. Product Description Preps

HighPrep HMW DNA Kit High molecular weight DNA extraction

HPHMW-D96 96
(96 Preps) size range of 50-300+ kb from whole
HighPrep HMW DNA Kit blood, saliva, buccal cells, cultured cells,
HPHMW-D96X4 tissues, and bacteria 384
(384 Preps)

Magnetic Separation Devices

Catalog No. Description

MYMAG-96 Handheld Magnetic Separation Device (96 well microplate format)

MYMAG-96X Magnetic Separation Device (96 well ring format)
MBMS-12 MagStrip Magnet Stand (1.5 mL x 12)
MBMS-31550 15 mL and 50 mL Magnetic Stand Combo (3 x 15 mL and 3 x 50 mL)

US/Canada: +1 301-302-0144 | Web: www.magbiogenomics.com | E-mail: [email protected]

Europe: +49 7250 33 13 403 | Web: www.magbiogenomics.com | E-mail: [email protected]

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www.magbiogenomics.com