Omega Mag-Bind M1378-00 User manual

Mag-Bind® Total Pure NGS
M1378-00 5 mL
M1378-01 50 mL
M1378-02 500 mL
August 2016

1
Introduction and Overview.......................................................2
Illustrated Protocol........................................................................3
Kit Contents and Preparations..................................................4
Storage and Stability...................................................................4
Mag-Bind® TotalPureNGS 96-wellPlateProtocol................5
Mag-Bind® Total Pure NGS 384-well Plate Protocol............8
Troubleshooting Guide.............................................................11
Ordering Information................................................................12
Manual Revision: August 2016
Innovations in nucleic acid isolation
Mag-Bind® Total Pure NGS
Table of Contents

2
Introduction and Principle
Omega Bio-tek’s Mag-Bind® Total Pure NGS Kit allows rapid and reliable isolation of PCR
products with high recovery rates. The system combines Omega Bio-tek’s proprietary
chemistries with the reversible nucleic acid-binding properties of magnetic beads to
selectively bind DNA fragments 100 bp and larger and eliminate excess nucleotides,
primers, and small, non-targeted amplication products, such as primer dimers. This kit
is designed for both manual and fully automated purication of PCR samples. Puried
PCR fragments can be used for microarrays, automated uorescent DNA sequencing,
restriction enzyme digestion, and other applications.
The Mag-Bind® Total Pure NGS magnetic particles technology provides a better solution
for nucleic acid purication compared to centrifugation and vacuum-based technologies.
The product can be easily scaled up while providing very user-friendly handling
procedures. If using Mag-Bind® Total Pure NGS for the rst time, please read this booklet
to become familiar with the procedures. DNA products are rst mixed and bind to Mag-
Bind® Total Pure NGS particles. With two rapid wash steps, trace contaminants such as
nucleotides, primers and small, non-targeted amplication products are removed. Pure
DNA is eluted in Elution Buer or water. Puried DNA can be directly used in downstream
applications without the need for further purication. The volume of Mag-Bind® Total Pure
NGS can be varied to selectivly bind dierence size fragments. Mag-Bind® Total Pure NGS
can be used with next generation sequencing library preparations protocols for reaction
clean-ups at amounts speciced by the library construction manufacturer.

3
Illustrated Protocol
Determine the Reaction size
Add Mag-Bind®Total Pure NGS and Mix
Magnetize and Remove Supernatant
Wash Twice with 70% Ethanol
Dry
Elute DNA

4
Kit Contents and Preparations
Storage and Stability
Mag-Bind® Total Pure NGS is guaranteed for at least 12 months from the date of purchase
when stored at 2-8°C.
Kit Contents
Product Number M1378-00 M1378-01 M1378-02
Mag-Bind® Total Pure NGS 5 mL 50 mL 500 mL
User Manual P P P

5
Mag-Bind® Total Pure NGS - 96-well Plate Protocol
Mag-Bind®Total Pure NGS Protocol for 96-well Plates
Materials and Equipment to be Supplied by User:
• 96-well PCR plate containing PCR samples (up to 50 μL/well)
• Magnetic separation device (Recommend AlpAqua Cat# 001322). For elution volumes
<30 µL AlpAqua Magnum FLX (Cat#A000400) is recommended.
• 96-well microplate or PCR Plate for elution
• Vortexer
• Multichannel pipettor
• Multichannel disposable reservoirs
• 70% ethanol
• Elution Buer (Cat#PDR048 or 10 mM Tris pH 8.0),TE Buer, or nuclease-free water
Before Starting:
• Bring the Mag-Bind® Total Pure NGS to room temperature before use.
1. Read the manufacturer’s instruction manual for the magnetic separation device, if
provided.
2. Place the 96-well PCR plate on the bench and measure the volume of the PCR
reaction. Determine the volume of Mag-Bind® Total Pure NGS that will be added to
the reaction. If the reaction volume will exceed 200 µL transfer to a microtiter plate
for processing.
Note: PCR reactions >20 µL will need to be transferred to a processing plate.
3. Shake or vortex the Mag-Bind® Total Pure NGS to resuspend any particles that may
have settled. Allow Mag-Bind® Total Pure NGS to come to room temperature before
use.
4. Add the desired volume of Mag-Bind® Total Pure NGS to each well based upon
desired fragment size to recover. Adding more Mag-Bind® Total Pure NGS binds
smaller fragments while using less will exclude smaller sizes. Volumes to add to
the sample is determined by the next generation sequencing library construction
instruction manual.
Example: 1.2X ratio required: 50 µL sample x 1.2 = add 60 µL Mag-Bind® Total Pure
NGS

6
5. Pipet up and down 5-10 times or vortex for 30 seconds.
6. Let sit at room temperature for 5 minutes.
7. Place the plate on a magnetic separation device to magnetize the Mag-Bind®
Total Pure NGS. Let sit at room temperature until the Mag-Bind® Total Pure NGS is
completely cleared from solution.
8. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind® Total
Pure NGS.
9. With the plate remaining on the magnet, add 200 μL 70% ethanol to each well.
10. Let sit at room temperature for 1 minute. It is not necessary to resuspend the Mag-
Bind® Total Pure NGS.
11. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind® Total
Pure NGS.
12. Repeat Steps 9-11 for a second 70% ethanol wash step.
13. Leave the plate on the magnetic separation device for 5-15 minutes to air dry the
Mag-Bind® Total Pure NGS. Remove any residual liquid with a pipettor.
Note: It is important to dry the Mag-Bind® Total Pure NGS before elution. Residual
ethanol may interfere with downstream applications.
Optional: Incubating the plate at 37°C can speed up evaporation.
14. Remove the plate from magnetic separation device.
15. Add 30-40 µL Elution Buer (not provided) to each well.
16. Pipet up and down 20 times or vortex for 30 seconds.
Mag-Bind® Total Pure NGS - 96-well Plate Protocol

7
17. Let sit at room temperature for 5 minutes.
18. Place the plate on a magnetic separation device to magnetize the Mag-Bind®
Total Pure NGS. Let sit at room temperature until the Mag-Bind® Total Pure NGS is
completely cleared from solution.
19. Transfer the cleared supernatant containing puried DNA to a new 96-well
microplate and seal with non-permeable sealing lm.
20. Store the plate at 2-8°C if storage is only for a few days. For long-term storage,
samples should be kept at -20°C.
Mag-Bind® Total Pure NGS - 96-well Plate Protocol

8
Mag-Bind® Total Pure NGS - 384-well Plate Protocol
Mag-Bind®Total Pure NGS Protocol for 384-well Plates
Materials and Equipment to be Supplied by User:
• 384-well PCR plate containing PCR samples (up to 100 μL/well)
• Magnetic separation device for 384-well PCR plates
• Skirted 384-well PCR plate
• Vortexer
• Multichannel pipettor
• Multichannel disposable reservoirs
• Sealing lm
• 70% ethanol
• Elution Buer (Cat#PDR048 or 10 mM Tris pH 8.0),TE Buer, or nuclease-free water
Before Starting:
• Bring the Mag-Bind® Total Pure NGS to room temperature before use.
1. Read the manufacturer’s instruction manual for the magnetic separation device, if
provided.
2. Place the 384-well PCR plate on the bench and measure the volume of the PCR
reaction. Transfer the sample to a skirted 384-well PCR plate.
3. Shake the Mag-Bind® Total Pure NGS to resuspend any Mag-Bind® Total Pure NGS
particles that may have settled. Allow Mag-Bind® Total Pure NGS to come to room
temperature before use.
4. Add the desired volume of Mag-Bind® Total Pure NGS to each well based upon
size of fragments to recover. Adding more Mag-Bind Total Pure NGS allows smaller
fragments to bind to magnetic beads while the larger fragments will continue to
bind. Volume to add to the sample is normaly determined by the next generation
sequencing library construction instruction manual.
Example: 1.2X ratio required: 50 µL sample x 1.2 = add 60 µL Mag-Bind® Total Pure
NGS

9
5. Pipet up and down 5-10 times or vortex for 30 seconds.
6. Let sit at room temperature for 5 minutes.
7. Place the plate on a magnetic separation device to magnetize the Mag-Bind®
Total Pure NGS. Let sit at room temperature until the Mag-Bind® Total Pure NGS is
completely cleared from solution.
8. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind® Total
Pure NGS.
9. Add 30 μL 70% ethanol to each well.
10. Let sit at room temperature for 1 minute. It is not necessary to resuspend the Mag-
Bind® Total Pure NGS.
11. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind® Total
Pure NGS.
12. Repeat Steps 9-11 for a second 70% ethanol wash step.
13. Leave the plate on the magnetic separation device for 5 minutes to air dry the Mag-
Bind® Total Pure NGS. Remove any residual liquid with a pipettor.
Note: It is important to dry the Mag-Bind® Total Pure NGS before elution. Residual
ethanol may interfere with downstream applications.
Optional: Incubating the plate at 37°C can speed up evaporation.
14. Remove the plate from magnetic separation device.
15. Add 30 µL Elution Buer (not provided) to each well.
16. Pipet up and down 20 times or vortex for 30 seconds.
Mag-Bind® Total Pure NGS - 384-well Plate Protocol

10
17. Let sit at room temperature for 2-3 minutes.
18. Place the plate on a magnetic separation device to magnetize the Mag-Bind®
Total Pure NGS. Let sit at room temperature until the Mag-Bind® Total Pure NGS is
completely cleared from solution.
19. Transfer the cleared supernatant containing puried DNA to a new 384-well
microplate and seal with non-permeable sealing lm.
20. Store the plate at 2-8°C if storage is only for a few days. For long-term storage,
samples should be kept at -20°C.
Mag-Bind® Total Pure NGS - 384-well Plate Protocol

11
Troubleshooting Guide
Please use this guide to troubleshoot any problems that may arise. For further assistance,
please contact the technical support sta, toll free, at 1-800-832-8896.
Possible Problems and Suggestions
Problem Cause Solution
Low yield
Low PCR product yield Increase the number amplication cycles
for PCR
Smaller PCR product size Small PCR fragments normally give lower
yield.
Ethanol residue During the drying step, remove any
liquid from bottom of the well.
Particle loss during the
procedure
Increase magnetization time.
Aspirate slowly.
DNA remains bound to
beads Increase elution volume.
Incomplete resuspension
of the beads during
elution
Vortex or pipet up and down to fully
resuspend the beads.
Problem Cause Solution
Primer
carryover
Insucient wash of the
particles
Wash the beads one more time with 70%
ethanol.
Problem Solution
Non-specic
amplication
products were
not removed
The size of the non-
specic amplication
products are larger than
100 bp
Non-specic amplication products
larger than 100 bp are not eciently
removed from PCR products.
Problem Cause Solution
Problems in
downstream
applications
Salt carryover 70% ethanol must be stored at room
temperature.
Ethanol carryover Ensure the beads are completely dried
before elution.

12
Ordering Information
The following components are available for purchase separately.
(Call Toll Free at 1-800-832-8896)
Product Part Number
Mag-Bind® Total Pure NGS (50 mL) M1378-01
Mag-Bind® Total Pure NGS (500 mL) M1378-02
Elution Buer (100 mL) PDR048
HiBind®, E.Z.N.A.®, Mag-Bind®, and MicroElute® are registered trademarks of Omega Bio-tek, Inc.
PCR is a patented process of Homan-La Roche. Use of the PCR process requires a license.