PurePrep Pathogens
Art.No.
OE00290096
Product Manual
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Tab le o f Co nt en ts
1. General Information ............................................................................................................ 3
1.1 Intended Use .................................................................................................................................... 3
1.2 Kit specifications ............................................................................................................................... 3
1.3 Basic principle ................................................................................................................................... 3
2. Materials ............................................................................................................................. 4
2.1 Kit Contents ..................................................................................................................................... 4
2.2 Reagents, consumables and equipment to be supplied by the user ........................................................... 5
3. Kit usage ............................................................................................................................. 6
3.1 Storage Conditions ............................................................................................................................ 6
3.2 Preparation of reagents ...................................................................................................................... 6
3.3 Safety instructions ............................................................................................................................. 6
3.4 Considerations .................................................................................................................................. 6
3.5 Magnetic Separation systems .............................................................................................................. 7
3.6 Shaker settings ................................................................................................................................. 7
3.7 Product use limitations ....................................................................................................................... 7
4. Protocols ............................................................................................................................. 8
4.1 Sample materials and pre-treatment procedures .................................................................................... 8
4.2 Manual processing of 200 µL liquid or pre-treated samples ...................................................................... 8
4.3 Protocol for the KingFisher Flex™ Magnetic Particle Processor .................................................................. 9
4.3.1 KingFisher BindIt software protocol .............................................................................................. 9
4.3.2 Preparation of processing plates ................................................................................................. 10
4.3.3 Detailed instructions ................................................................................................................. 10
5. Troubleshooting ................................................................................................................ 12
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1. General Information
1.1 Intended Use
PurePrep Pathogens is intended for Research Use Only (RUO). The kit is suited for qualified personnel only.
The kit is intended for manual and automated isolation of nucleic acids (DNA and RNA) from a wide range of
samples. Processing time for the preparation of 96 samples is about 40 minutes. The kit requires no
phenol/chloroform extraction or alcohol precipitation and eliminates the need for repeated centrifugation, vacuum
filtration or column separation. It allows safe handling of potentially infectious samples and is designed to avoid
sample-to-sample cross-contaminations. The obtained nucleic acids can be used directly as template for
downstream applications such as PCR, qPCR, qRT-PCR or any kind of enzymatic reaction.
PurePrep Pathogens is suitable for use with blood samples, liquid samples (e.g. plasma, serum, urine, swab
washes), tissue samples, feces. For details on the individual procedures for sample pre-treatment see below.
PurePrep Pathogens magnetic beads are optimized for use in isolating total nucleic acids. The beads are easy to
handle and are supplied in an optimized storage buffer for increased suspension time.
1.2 Kit specifications
The kit provides reagents for extraction of total nucleic acids from 200 µL liquid sample or 200 µL homogenized
tissue samples, cells or suspended feces. Total nucleic acids are finally eluted in a volume of 100 µL Elution Buffer.
The obtained nucleic acids should be used for qPCR, qRT-PCR immediately after extraction. Storage at <-20°C is
recommended for later analysis.
1.3 Basic principle
Samples are lysed under denaturing conditions by adding Lysis Buffer PA1 and Proteinase K. During the incubation
the released nucleic acids can bind to the PurePrep magnetic beads. After magnetic separation and discard of the
supernatant, the beads are washed three times using alcoholic buffers (Wash Buffers I and II) to remove
contaminants and salts. A drying step makes sure all traces of ethanol from the final wash steps are removed.
Finally, purified nucleic acids are eluted with low-salt Elution Buffer and can directly be used for downstream
applications.
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2. Materials
96 preps
MDKT00210096
Lysis Buffer P A1
20 mL
Binding Buffer U1
40 mL
Wash B uffer I
2 x 80 mL
Wash B uffer II
80 mL
Elution Buffer
20 mL
Proteinase K
20 mg
(for 1 mL working solution)
Poly-A-RNA
0.3 mg
Poly-A-RNA Buffer
0.5 mL
PurePrep
2 mL
2.1 Kit Contents
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2.2 Reagents, consumables and equipment to be supplied by the user
Protocol
Manual use
Automated use
Sample containers
1.5 or 2 mL microtubes
Recommended:
Riplate®SW 96, PP, 2 mL, (Ritter, 43001–0020)
Nunc™ 96-well Polypropylene DeepWell™
Storage Plate 2.0 mL, (Thermo Scientific,
Art.No. 278752)
Magnetic separation
MM-Separator M12 + 12 P
Art.No. MDMG0001
MM-Separator 96 DeepWell
Art.No. MDMG0013
Final container
1.5 or 2 mL microtubes
96-well microplate
Mixing
Tube Vo rtex er
Microplate shaker (min. 1000 RPM)
Product
Art. No.
Contents
2 mL DeepWell Plate with
square wells for
KingFisher™
MDPL00200060
60 pieces
200 µL square-well Elution
Plate for KingFisher™
MDPL00190060
60 pieces
96 well Tip-Comb for
KingFisher™
MDPL00210060
60 pieces
Reagents:
• molecular biology grade (nuclease free) water to reconstitute Proteinase K
Consumables/equipment:
Consumables for processing on the KingFisherTM Flex instrument
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3. Kit usage
3.1 Storage Conditions
All kit components including Proteinase K (lyophilized), Poly-A-RNA (lyophilized) and PurePrep can be stored at
room temperature. Store ready solutions of Proteinase K and Poly-A-RNA in suitable aliquots at -20°C. When stored
under the conditions mentioned, the kit is stable as indicated by the expiry date on the label.
3.2 Preparation of reagents
• Reconstitute Proteinase K:
MDKT00210096 (96 preps), add 1 mL of molecular biology grade water to the vial of Proteinase K
and vortex to dissolve. Store solution of Proteinase K in aliquots at -20°C.
• Reconstitute Poly-A-RNA:
Add 120 µL of Poly-A-RNA Buffer to each vial of Poly-A-RNA and vortex to dissolve. Store
solutions of Poly-A-RNA in aliquots at -20°C.
• If there is any precipitate present in the buffers, warm the buffer to 25-37°C to dissolve the precipitate
before use.
• Immediately before use, resuspend PurePrep beads by vortexing for 20 seconds.
• Samples should be thoroughly mixed before use.
3.3 Safety instructions
Tak e ap pr op r ia te s a fe ty me as ur es , s uc h a s we ar in g a s ui t ab le l a b co at , d is po s ab le g l ov e s, a nd pr ot ec ti v e g og gl es .
Follow local legal requirements for working with biological materials.
More information is found in the safety data sheets (SDS), available at www.molg3n.com
Infectious potential of liquid waste left over after using the PurePrep Pathogens kit was not tested. Even though
contamination of waste with residual infectious material is unlikely, it cannot be excluded completely. Therefore,
liquid waste should be handled as being potentially infectious, and discarded according to local safety regulations.
3.4 Considerations
1. To a v o id c r o ss -contamination and DNA degradation, change pipette tips after each use and use nucleasefree filter-tips.
2. Avoid leaving bottles open to prevent contamination or evaporation of the kit reagents.
3. Do not combine components of different kits unless the lot numbers are identical.
4. Process only as many samples in parallel as the magnetic separator allows.
5. The elution can be done in smaller volumes of Elution Buffer. Although this may result in higher nucleic
acids concentrations, overall yield may be lower. The yield may also be increased by prolonging the
incubation time, and with pre-heated Elution Buffer (56°C).
6. The Elution Buffer does not contain EDTA.
7. Avoid samples containing coagulates or precipitates, as this may result in poor results or quality.
Centrifuge samples before use.
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8. The kit is compatible with whole blood treated with EDTA and citrate. Heparin is co-isolated and may
interfere with subsequent PCR analyses.
9. It may occur that a small amount of beads is accidentally transferred with the final DNA sample, but most
likely this will not inhibit subsequent applications. However, if desired another separation step can be
performed to remove the beads.
3.5 Magnetic Separation systems
PurePrep Pathogens has been designed for use on the MM-Separator 96 DeepWell and MM-Separator M12 + 12 P.
The MM-Separator M12 + 12 P (Art. No. MDMG0001) allows simultaneous processing of up to 12 samples in 2 mL
microcentrifuge tubes. For processing in 96 DeepWell plates, use the MM-Separator 96 DeepWell (Art.No.
MDMG0013).
For use with other magnetic separators, please contact the technical support at support@molg3n.com.
PurePrep Pathogens is compatible with KingFisher™ Flex Magnetic Particle Processor by Thermo Scientific™.
Protocols and consumables are available on request.
3.6 Shaker settings
The speed settings for the microplate shaker described in the protocols that follow were defined with a specific
instrument and microplate. When first using a plate shaker for incubation steps, the speed settings have to be set
carefully for each specific plate to prevent cross contamination and spillage. Setting the speed of the shaker can be
done by loading a microplate with a volume of dyed water equal to the working volume during each step, and stepwise increasing the shaker speed until droplets are observed on the surface of the plate. Set the shaker speed lower
again.
3.7 Product use limitations
PurePrep Pathogens is intended for Research Use Only. Do not use for other purposes than intended. The kit
components can be used only once.
No guarantee is offered when using sample material other than specified. It is recommended to check the suitability
of the purified nucleic acids for each selected qPCR / qRT-PCR assay.
The end-user has to validate the performance of the kit for any particular use, since the performance characteristics
of the kits have not been validated for any specific application. Molgen kits may be used in clinical diagnostic
laboratory systems after the laboratory has validated the complete diagnostic system as required by CLIA’ 88
regulations in the U.S. or equivalents in other countries.
The product is intended for use by trained personnel. The isolated nucleic acids can be used in most genomic
applications, such as PCR, qPCR.
Diagnostic results generated using the sample preparation procedure should only be interpreted with regard to
other clinical or laboratory findings. Adequate controls should be used in each set of isolations, especially when used
for diagnostic purposes.
4. Protocols
4.1 Sample materials and pre-treatment procedures
Preparation of the Lysis Master Mix
For each sample mix 200 µL Lysis Buffer PA1 with 1 µL reconstituted Poly-A-RNA and 10 µL of reconstituted
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Proteinase K solution (20 mg/mL). Prepare an excess of the Lysis Master Mix to compensate for pipetting inaccuracy
Sample material
Pre-treatment procedure
Further processing
Liquid samples (e.g.
plasma, serum, blood,
swab wash solutions)
n/a
Use up to 200 µL of the liquid sample and add
211 µL Lysis Master Mix
Dry swabs without
transport media
Add an appropriate volume of molecular
biology grade water to dried swab (swab
should be completely submerged) and
shake/vortex vigorously for 15 minutes.
Take 2 0 0 µ L f or f u rt h er p r oc es s in g
Use 200 µL of the wash solution and add 211 µL
Lysis Master Mix
Feces / stool
Add 1.5 mL molecular biology grade water
to a pea-size amount of feces. Mix well by
vortexing. Spin down at low g-forces to
remove remaining particulate sample
residuals. Use 200 µL of the suspension for
further processing.
Use up to 200 µL of the centrifuged suspension
and add 211 µL Lysis Master Mix
Tissue samples
Mechanically homogenize <30 mg of tissue
sample in 500 µL molecular biology grade
water using suitable devices (bead beater).
Spin down for 1 min at 8,000 x g to remove
debris. Use 200 µL of the suspension for
further processing.
Use up to 200 µL of the centrifuged suspension
and add 211 µL Lysis Master Mix
especially when using multichannel pipettes etc. Use the Lysis Master Mix immediately after preparation.
Recommendations for sample pre-treatment
For samples not mentioned in the table above please contact Molgen for support protocols at support@molg3n.com.
4.2 Manual processing of 200 µL liquid or pre-treated samples
Before starting:
• Check if Lysis Master Mix was prepared according to section 4.1.
• Pre-treat of samples (if required) according to section 4.1
• Vortex magnetic beads thoroughly into a homogeneous suspension.
This protocol is intended for manual use of the kit. It can also be used as a guideline to set up an automated
procedure on liquid handling instruments. For this suitable 96-well plates and accessories can be used. Make sure
that the liquid handling devices is equipped with the required devices (shaker, incubator, magnetic separator etc.).
1. Tran s f e r 200 µL sample into a microtube. If the volume is lower than 200 µL, bring the volume up to 200
µL with 1 x PBS buffer or molecular biology grade water.
2. Add 211 µL Lysis Master Mix. Mix immediately and incubate on a shaker for 10 min with shaking at
1000 RPM. Spin down briefly to collect any sample from the microtube lid.
3. Add 20 µL PurePrep beads and 400 µL Binding Buffer U1 . Mix and incubate on a shaker for 5 min
with shaking at 1000 RPM. Spin down briefly to collect any sample from the microtube lid.
4. Place the samples on the magnetic separator and wait at least 1 minute to collect the beads. Remove
supernatants without disturbing the attracted magnetic bead pellet.
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5. Remove the sample plate from the magnetic separator and add 800 µL Wash Buffer I to the tubes.
Resuspend the beads by incubation of the samples on a shaker for 1 min at 1000 RPM (alternatively
pipette the wash buffer up and down until the beads are resuspended completely). Place the samples in a
magnetic separator and wait at least 1 minute to collect the beads. Remove the supernatants without
disturbing the attracted bead pellet.
6. Repeat step 5 one more time with 800 µL Wash Buffer I and one more time with 800 µL Wash
Buffer II .
7. Dry the beads on air for 10 min to evaporate the ethanol completely. If necessary. briefly spin down and
remove any buffer residues before drying the attracted magnetic beads.
8. Remove the samples from the magnetic separator and add 100 µL Elution Buffer . Resuspend the
attracted magnetic beads by repeated pipetting up and down. Incubate on a shaker for 10 min at 1000
RPM.
9. Place the tubes in a magnetic separator and wait at least 1 minute to collect the beads. Transfer the
eluates to new tubes. The purified nucleic acids in the eluate are now ready to use.
• If the transferred eluates appear turbid, briefly centrifuge the samples and carefully transfer the
eluates.
• If the transferred eluates contain magnetic particles, place the tubes on the magnetic separator again,
separate for 1 minute and transfer the eluates.
• The nucleic acids can be eluted with a lower volume of Elution Buffer (depending on the expected
yield). The minimum volume for elution is 30 µL and this can reduce the yield. If a large amount of
nucleic acids is expected, the volume of Elution Buffer can be increased.
4.3 Protocol for the KingFisher Flex™ Magnetic Particle Processor
4.3.1 KingFisher BindIt software protocol
Please contact Molgen for the most recent BindIt software method files. We provide the corresponding files for
direct upload on the KingFisherTM magnetic particle processors. A PDF description of the method file is included.
Refer to the BindIt software manual regarding the upload procedure of the supplied software files to the instrument.
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4.3.2 Preparation of processing plates
Plate
Type*)
Reagent
Volume
Sample Plate
2 mL DeepWell Plate with square wells
for KingFisher™
(pre-treated) sample adjusted
to 200 µL
Lysis Master Mix
PurePrep beads
Binding Buffer U1
200 µL
211 µL
20 µL
400 µL
Wash B uffer I 1st
2 mL DeepWell Plate with square wells
for KingFisher™
Wash B uffer I
800 µL
Wash B uffer I 2nd
2 mL DeepWell Plate with square wells
for KingFisher™
Wash B uffer I
800 µL
Wash B uffer II
2 mL DeepWell Plate with square wells
for KingFisher™
Wash B uffer II
800 µL
Elution Buffer
200 µL square-well Elution Plate for
KingFisher™
Elution Buffer
100 µL
Tip plate
2 mL DeepWell Plate with square wells
for KingFisher™
Empty, for loading Tip-Comb
only
N/A
Initial plate filling for instrument set-up:
To b e a d de d a ft e r t he i n i ti a l l ys i s s te p o n t h e Ki n g F i sh e r
TM
Flex magnetic particle processor
*) Suitable plates can be purchased at Molgen (see section 2.2). We strongly recommend using only the plates
which are intended to use on the KingFisherTM magnetic particle processor. Using unsuitable plates may result in
extraction failure or instrument damage.
4.3.3 Detailed instructions
Follow exactly the instructions as given below. Do not change the order of reagent addition for the Sample
Plate. Label all plates thoroughly and unambiguously to avoid any misloading during the instrument loading
procedure.
1. Prepare samples according to section 4.1. Add 200 µL of the sample to each well of the Sample Plate. If
the volume is lower than 200 µL, bring the volume up to 200 µL with 1 x PBS buffer or molecular biology
grade water.
2. Add 211 µL of Lysis Master Mix to each well of the Sample Plate. Mix by pipetting up and down until a
homogeneous mixture is visible (typically 3 – 4 times). Incubate on a suitable plate shaker for 10 min with
shaking at 1000 rpm. Continue with preparing the remaining plates before continuing with the sample
plate.
3. Prepare two plates for the 1
each well of the corresponding deep-well plates.
st
and 2nd wash steps with Wash Buffer I. Add 800 µL Wash Buffer I to
4. Prepare one plates for the 3rd wash step with Wash Buffer II. Add 800 µL Wash Buffer II to each
well of the corresponding deep-well plate.
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5. Prepare one plate for Elution Buffer. Add 100 µL Elution Buffer to each well of the corresponding
plate.
6. Add 20 µL of PurePrep beads to each well of the Sample Plate.
7. Add 400 µL of Binding Buffer U1 to each well of the Sample Plate.
8. Switch on the KingFisherTM Flex magnetic particle processor and select the “PurePrep Pathogens
PostLysis” protocol from the user defined protocols
9. Start the protocol.
10. Load the plates to the instrument, following the instructions on the instrument display. Order of plates start
with the tip plate and ends with the sample plate. The purification process starts immediately after loading
the sample plate to the instrument.
Make sure that all plates are inserted in the same orientation (especially when using partially filled plates). Place
the A1 well of each plate to the A1 mark on the instruments turntable.
11. At the end of the method remove all plates from the instrument. Follow the information on the instrument
display.
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5. Troubleshooting
Problem
Possible causes
Comments and suggestions
Low nucleic acid
yield
Insufficient sample lysis
- Optimize sample pre-treatment, make sure that Lysis
Master Mix includes Proteinase K
Inefficient binding to the magnetic
particles
- Use correct amount of all reagents
- Increase mixing steps incubation time for binding step
- Mix sample during lysis / binding incubation
Insufficient washing procedure
- Make sure that beads are completely resuspended in the
washing steps.
Incomplete elution
- Drying of Wash Buffer II may have been incomplete
- Completely resuspend the beads in the elution step.
Problems in
downstream
applications /
contamination in
DNA sample
Ethanol in the eluted DNA
- Increase the drying time to 15 minutes
Salt in the eluate
- Make sure that all supernatants are properly removed.
- Avoid carry-over of Lysis Master Mix, Binding Buffer or
Wash B uffer s to th e elu ate.
Magnetic beads remaining in the
eluate
- Place the tubes with eluates in the magnetic separator
again and transfer the supernatant to a new container.
High amounts of co-purified genomic DNA (e.g. for cell and
tissue samples) may cause high viscosity of the eluate and
force incomplete bead separation in the elution step. Use
higher volume of elution buffer and/or reduce sample
input.
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