Macherey-Nagel NucleoMag 96 PCR User manual

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DE Tel.: [phone] [email protected]
CH Tel.: [phone] [email protected]
FR Tel.: [phone] [email protected]
US Tel.: [phone] [email protected]
MACHEREY-NAGEL GmbH & Co. KG
Valencienner Str. 11
52355 Düren · Deutschland
Bioanalysis
MACHEREY-NAGEL
User manual
June 2021 / Rev. 04
nNucleoMag®96 PCR
PCR clean-up

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Tel.: [phone]
Toll-free: 0800 26 16 000 (Germany only)
E-mail: [email protected]
Technical Support Bioanalysis
Tel.: [phone]
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3MACHEREY-NAGEL – 06/2021, Rev. 04
PCR clean-up
Table of contents
1 Components 4
1.1 Kit contents 4
1.2 Equipment and consumables to be supplied by user 4
1.3 About this user manual 4
2 Product description 5
2.1 The basic principle 5
2.2 Kit specifications 5
2.3 Magnetic separation systems 5
2.4 Adjusting the shaker settings 6
2.5 Handling of beads 6
2.6 Elution procedures 7
3 Storage conditions and preparation of working solutions 8
4 Safety instructions 9
5 Protocol for clean-up of 50 μL PCR reactions 10
6 Appendix 14
6.1 Troubleshooting 14
6.2 Ordering information 15
6.3 Product use restriction / warranty 16

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1 Components
1.1 Kit contents
NucleoMag®96 PCR
REF
1 x 96 preps
744100.1
4 x 96 preps
744100.4
24 x 96 preps
744100.24
NucleoMag®P-Beads 1.4 mL 5.6 mL 33.6 mL
Binding Buffer MP1 20 mL 80 mL 500 mL
Wash Buffer MP2 40 mL 160 mL 1000 mL
Wash Buffer MP3 (Concentrate)* 25 mL 100 mL 2 x 200 mL
Elution Buffer MP4 15 mL 30 mL 250 mL
Elution Plate U-bottom
(including Self-adhering PE Foil)
1 4 24
User manual 1 1 1
1.2 Equipment and consumables to be supplied by user
Product REF Pack of
• Magnetic separation system
e.g., NucleoMag®SEP (see section 2.3)
744900 1
• Separation plate for magnetic beads
separation,
e.g., Square-well Block (96-well block with
2.1 mL square-wells)
740481
740481.24
4
24
1.3 About this user manual
It is recommended to read the instructions of this user manual carefully before use. All
technical literature is also available on the internet at www.mn-net.com.
Please contact Technical Service regarding information about changes of the current user
manual compared to previous or updated revisions.
¹ For preparation of working solutions and storage conditions see section 3.

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2 Product description
2.1 The basic principle
The NucleoMag®96 PCR procedure is based on reversible adsorption of nucleic acids to
paramagnetic beads under appropriate buffer conditions. Adjusting the PCR reaction to
binding conditions and addition of paramagnetic beads can be carried out simultaneously.
After magnetic separation and removal of supernatant, the beads are washed to remove
contaminants and salt. A short drying step is necessary to remove ethanol from previous
washing steps. Finally, highly purified DNA is eluted with low salt elution buffer or water and
can be used directly for downstream applications. The NucleoMag®96 PCR kit can be used
either manually or automated on standard liquid handling instruments.
2.2 Kit specifications
• NucleoMag®96 PCR is designed for rapid manual and automated clean-up of PCR
fragments using the NucleoMag®SEP Magnetic Separator (see ordering information)
other magnetic separation systems (see section 2.3). Manual processing time for 96
samples is about 45 min.
• NucleoMag®96 PCR is easily adapted to common liquid handling instruments.
The actual processing time and sample volume to be processed depends on the
configuration of your instrument and the used magnetic separation system. Typically, 96
samples can be purified in about 30–45 min.
• The kit provides reagents for the purification of 3–5 μg DNA from 50 μL PCR samples.
• Typical concentration of the purified PCR samples of 75–200 ng/μL can be achieved.
• The purity of recovered PCR products is A260/A280 ≥ 1.7–1.9.
• The kit is designed for use with or without detergent containing PCR buffers.
• Purified PCR products are ready-to-use for downstream applications, like automated
fluorescent sequencing, labeling, microarray analysis, cloning, or restriction digestion.
• NucleoMag®96 PCR can be processed completely at room temperature. Elution at
55 °C will increase the recovery by about 10–15 %.
• NucleoMag®P-Beads are highly reactive, superparamagnetic beads. The binding
capacity is 0.3 μg of DNA per 1 μL of NucleoMag®P-Bead suspension, 1 μL of
suspension contains 150 μg of beads.
2.3 Magnetic separation systems
For use of NucleoMag®96 PCR, the use of the magnetic separator NucleoMag®SEP is
recommended. Separation is carried out in a Square-well Block (see ordering information).
The kit can also be used with other common separators.
Magnetic separator Separation plate or tube
NucleoMag®SEP (MN REF 744900) Square-well Block (MN REF 740481)
Tecan Te-MagS™ 1.5 mL tubes without lid (Sarstedt)

MACHEREY-NAGEL – 06/2021, Rev. 046
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Static magnetic pins
Separators with static magnetic pins, for example, NucleoMag®SEP (for manual use and for
use on liquid handling workstations): This type of separator is recommended in combination
with a suitable microplate shaker for optimal resuspension of the beads during the washing
and elution steps. Alternatively, beads can be resuspended in the buffer by pipetting up and
down several times. For fully-automated use on liquid handling workstations, a gripper tool is
required, the plate is transferred to the magnetic separator for separation of the beads and
transferred to the shaker module for resuspension of the beads.
Movable magnetic systems
Separators with moving magnetic pins: Magnetic pins/rods are moved from one side of the
well to the other and vice versa. Beads follow this movement and are thus pulled through
the buffer during the wash and elution steps. Separation takes place when the system stops.
Automated separators
Separators with moving magnets: Magnetic beads are transferred into suitable plates or
tubes. Beads are resuspended from the rod-covered magnets. Following binding, washing or
elution beads are collected again with the rod-covered magnets and transferred to the next
plate or tube.
2.4 Adjusting the shaker settings
When using a plate shaker for the washing and elution steps, the speed settings have
to be adjusted carefully for each specific separation plate and shaker to prevent cross-
contamination from well to well. Proceed as follows:
Adjusting shaker speed for binding and wash steps:
• Load 300 μL dyed water (select to the wells of the separation plate. Place the plate on
the shaker and start shaking with a moderate speed setting for 30 seconds. Turn off the
shaker and check the plate surface for small droplets of dyed water.
• Increase speed setting, shake for an additional 30 seconds, and check the plate
surface for droplets again.
• Continue increasing the speed setting until you observe droplets on top of the
separation plate. Reduce speed setting, check again, and use this setting for the
washing step.
Adjusting shaker speed for the elution step:
• Load 100 μL dyed water to the wells of the collection plate and proceed as described
above.
2.5 Handling of beads
Distribution of beads
A homogeneous distribution of the magnetic beads to the individual wells of the separation
plate is essential for a high well-to-well consistency. Therefore, before distributing the beads,
make sure that the beads are completely resuspended. Shake the storage bottle well or
place it on a vortexer shortly. Premixing magnetic beads with the binding buffer allows easier
homogenous distribution of the beads to the individual wells of the separation plate. During

7MACHEREY-NAGEL – 06/2021, Rev. 04
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automation, a premix step before aspirating the beads/binding buffer mixture from the
reservoir is recommended to keep the beads resuspended.
Magnetic separation time
Attraction of the magnetic beads to the magnetic pins depends on the magnetic strength of
the magnetic pins, the selected separation plate, distance of the separation plate from the
magnetic pins, and the volume to be processed. The individual times for complete attraction
of the beads to the magnetic pins should be checked and adjusted on each system. It is
recommended using the separation plates or tubes specified by the supplier of the magnetic
separator.
Washing the beads
Washing the beads can be achieved by shaking or mixing. In contrast to mixing by pipetting
up and down, mixing by shaker or magnetic mixing allows simultaneous mixing of all samples.
This reduces the time and number of tips needed for the preparation. Resuspension by
pipetting up and down, however, is more efficient than mixing by a shaker or magnetic mix.
Method Resuspension
efficiency
Speed Number of tips
needed
Magnetic mix + ++ Low
Shaker ++ ++ Low
Pipetting +++ +* High
+: acceptable, ++: good, +++: excellent, * 8-channel pipetting device
2.6 Elution procedures
Purified DNA can be eluted directly with the supplied Elution Buffer MP4 or water (pH7.5–8.5).
Elution can be carried out in a volume of ≥ 25 μL (per 12 μL bead suspension). It is essential
to cover the NucleoMag®P-Beads completely with elution buffer during the elution step. The
volume of dispensed elution buffer depends on the magnetic separation system (e.g., the
position of the pellet inside the separation plate). For efficient elution, the magnetic bead
pellet should be resuspended completely in the elution buffer. For some separators, high
elution volumes might be necessary to cover the whole pellet.
Elution is possible at room temperature. Yield can be increased by 10–15 % if elution is
performed at 55 °C.

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3 Storage conditions and preparation of working
solutions
Attention: Buffers MP1 and MP2 contain chaotropic salt! Wear gloves and goggles!
CAUTION: Buffers MP1 and MP2 contain guanidine hydrochloride which can form highly
reactive compounds when combined with bleach (sodium hypochlorite). DO NOT add bleach
or acidic solutions directly to the sample-preparation waste.
Storage conditions:
• All components of the NucleoMag®96PCR kit should be stored at room temperature
(15–25 °C) and are stable for up to one year.
Before starting any NucleoMag®96 PCR protocol, prepare the following:
• Wash Buffer MP3: Add the indicated volume of ethanol (96–100 %) to Buffer MP3
Concentrate. Mark the label of the bottle to indicate that ethanol was added.
Wash Buffer MP3 is stable at room temperature (15–25 °C) for at least one year.
NucleoMag®96 PCR
REF 1 x 96 preps
744100.1
4 x 96 preps
744100.4
24 x 96 preps
744100.24
Buffer MP3
(Concentrate)
25 mL
Add 100 mL ethanol
100 mL
Add 400 mL ethanol
2 x 200 mL
Add 800 mL ethanol
to each vial

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4 Safety instructions
The following components of the NucleoMag®96 PCR kits contain hazardous contents.
Wear gloves and goggles and follow the safety instructions given in this section.
Component Hazard contents GHS
symbol
Hazard
phrases
Precaution
phrases
Inhalt Gefahrstoff GHS-Symbol H-Sätze P-Sätze
MP1 guanidine hydrochloride
30–50 %
Guanidinhydrochlorid 36–50 %
CAS 50-01-1 WARNING
ACHTUNG
302, 319 264W, 280sh,
301+312, 330
MP2 guanidine hydrochloride
24–36 %
Guanidinhydrochlorid 24–36 %
CAS 50-01-1 WARNING
ACHTUNG
302 264W, 301+312,
330
The symbol shown on labels refers to further safety information in this section.
Das auf Etiketten dargestellte Symbol weist auf weitere Sicherheitsinformationen dieses Kapitels hin.
Hazard phrases
H 302 Harmful if swallowed.
Gesundheitsschädlich bei Verschlucken.
H 319 Causes serious eye irritation.
Verursacht schwere Augenreizung.
Hazard phrases
P 264W Wash with water thoroughly after handling
Nach Gebrauch mit Wasser gründlich waschen.
P 280sh Wear protective gloves / eye protection.
Schutzhandschuhe / Augenschutz tragen.
P 302 IF SWALLOWED: Call a POISON CENTER/doctor if you feel unwell.
BEI VERSCHLUCKEN: Bei Unwohlsein GIFTINFORMATIONSZENTRUM/Arzt anrufen.
P 330 Rinse mouth.
Mund ausspülen.
For further information please see Material Safety Data Sheets (www.mn-net.com).
Weiterführende Informationen finden Sie in den Sicherheitsdatenblättern (www.mn-net.com).

MACHEREY-NAGEL – 06/2021, Rev. 0410
PCR clean-up
5 Protocol for clean-up of 50 μL PCR reactions
Protocol-at-a-glance
• For additional equipment and hardware requirements, refer to section 1.2 and 2.3,
respectively.
• For detailed information on each step, see page 12.
Before starting the preparation:
• Check if Buffer MP3 was prepared according to section 3.
1 Transfer PCR reaction
mixture to appropriate
96-well plate
50 μL PCR reaction
For PCR reactions < 50 μL, adjust
the volume to 50 μL
using sterile water
2 Bind DNA to
NucleoMag®P-Beads
12 μL NucleoMag®P-Beads
138 μL MP1
Mix by shaking
for 5 min at RT
(Optional: Mix by pipetting
up and down)
Remove supernatant
after 2 min separation
3 Wash with MP2 Remove Square-well Block
from NucleoMag®SEP
300 μL MP2
Resuspend: Shake 5 min at RT
(Optional: Mix by pipetting
up and down)
Remove supernatant
after 2 min separation