Protocol
PrepSEQ™ Nucleic Acid Extraction Kit
© Copyright 2009, 2010 Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
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All other trademarks are the sole property of their respective owners.
Part Number 4400739 Rev. C
06/2010
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
How to use this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
How to obtain support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
Chapter 1
PrepSEQ™ Nucleic Acid Extraction Kit
for Pharmaceutical Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Preparation and extraction of lysate DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Chapter 2
PrepSEQ™ Nucleic Acid Extraction Kit
for Food Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Preparation and extraction of lysate DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Appendix A
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Chemical waste safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Chemical alerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
iii
Contents
iv
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Preface
Preface
Safety information
Note: For general safety information, see this Preface and Appendix A, “Safety”
on page 25. When a hazard symbol and hazard type appear by a chemical name or
instrument hazard, see the “Safety” Appendix for the complete alert on the
chemical or instrument.
Safety alert words
Four safety alert words appear in Applied Biosystems user documentation at
points in the document where you need to be aware of relevant hazards. Each alert
word—IMPORTANT, CAUTION, WARNING, DANGER—implies a
particular level of observation or action, as defined below:
IMPORTANT! – Indicates information that is necessary for proper instrument
operation, accurate chemistry kit use, or safe use of a chemical.
CAUTION! – Indicates a potentially hazardous situation that, if not
avoided, may result in minor or moderate injury. It may also be used to alert
against unsafe practices.
WARNING! – Indicates a potentially hazardous situation that, if not
avoided, could result in death or serious injury.
DANGER! – Indicates an imminently hazardous situation that, if not
avoided, will result in death or serious injury. This signal word is to be
limited to the most extreme situations.
MSDSs
The MSDSs for any chemicals supplied by Applied Biosystems or Ambion are
available to you free 24 hours a day. For instructions on obtaining MSDSs, see
Appendix A.
IMPORTANT! For the MSDSs of chemicals not distributed by
Applied Biosystems or Ambion contact the chemical manufacturer.
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
v
Preface
How to use this guide
How to use this guide
Text conventions
This guide uses the following conventions:
• Bold text indicates user action. For example:
Type 0, then press Enter for each of the remaining fields.
• Italic text indicates new or important words and is also used for emphasis.
For example:
Before analyzing, always prepare fresh matrix.
• A right arrow symbol () separates successive commands you select from a
drop-down or shortcut menu. For example:
Select FileOpenSpot Set.
Right-click the sample row, then select View Filter View All Runs.
User attention words
Two user attention words appear in Applied Biosystems user documentation. Each
word implies a particular level of observation or action as described below:
Note: – Provides information that may be of interest or help but is not critical to
the use of the product.
IMPORTANT! – Provides information that is necessary for proper instrument
operation, accurate chemistry kit use, or safe use of a chemical.
How to obtain support
For the latest services and support information for all locations, go to
www.appliedbiosystems.com.
At the Applied Biosystems web site, you can:
• Access worldwide telephone and fax numbers to contact Applied Biosystems
Technical Support and Sales facilities
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order Applied Biosystems user documents, MSDSs, certificates of analysis,
and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
vi
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 1
Product overview
The PrepSEQ™ Nucleic Acid Extraction Kit provides a simple way to prepare
DNA from broth cultures.
Required materials
Kit contents
The PrepSEQ™ Nucleic Acid Extraction Kit (PN 4400799) contains reagents for
100 reactions. Kit components are shown in the table below. For information on
the kit contents, refer to the “Materials supplied” section in the packaging insert
for your kit.
Item
Quantity or
volume
Lysis Buffer, 2 bottles
50 mL/bottle
Magnetic Particles, 2 tubes
1.5 mL/tube
Binding Solution (Isopropanol), 1 empty bottle
NA
Wash Buffer Concentrate, 2 bottles
26 mL/bottle
Elution Buffer, 1 bottle
25 mL
Proteinase K (PK) Buffer, 1 bottle
50 mL
Proteinase K, 1 tube
1.25 mL
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
1
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Pharmaceutical Testing
PrepSEQ™ Nucleic Acid Extraction Kit
for Pharmaceutical Testing
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Required materials
Storage
For the following hazards, see the complete safety alert descriptions in
Appendix A on page 29:
DANGER! CHEMICAL HAZARD. Lysis Buffer.
WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate.
WARNING! CHEMICAL HAZARD. Elution Buffer.
WARNING! CHEMICAL HAZARD. Proteinase K Buffer.
WARNING! CHEMICAL HAZARD. Proteinase K.
WARNING! CHEMICAL HAZARD. Magnetic Particles.
• Room temperature:
– Lysis Buffer
– Binding Solution – Before its use for the first-time, add 30 mL of 100%
isopropanol to the empty Binding Solution bottle. Label the bottle to
indicate that isopropanol is added.
– Wash Buffer – Before its use for the first-time, add 74 mL of 95%
ethanol to the Wash Buffer Concentrate bottle, mix well, then label the
bottle to indicate that ethanol is added.
– Elution Buffer
– Proteinase K Buffer
• Magnetic Particles: 2 to 8 °C
IMPORTANT! White precipitate occasionally forms in the magnetic particles
tube. Extraction experiments show that formation of precipitate does not
affect performance. If precipitate forms, incubate the tube at 37 °C for 10
minutes, then vortex to completely resuspend the particles.
• Proteinase K: –20 °C
For information on storage of kit components, refer to the “Storage” section in the
packaging insert for your kit.
2
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Required materials
The following table includes materials for using (but not included in) the
PrepSEQ™ Nucleic Acid Extraction Kit. Unless otherwise indicated, many of the
listed items are available from major laboratory suppliers (MLS).
Equipment, consumables, and reagents
Item
Source
Equipment
Two block heaters, for use with 2-mL tubes,
56 °C and 70 °C
MLS
Note: If only one block heater is available, set
temperature to 56 °C then reset to 70 °C after the
56 °C incubation. If two block heaters are
available, set one to 56 °C and the other to 70 °C.
Ice bucket
MLS
Magnetic stand, 6-tube
AM10055
Refrigerated benchtop microcentrifuge for 1.5and 2-mL tubes, 2 to 8 °C
MLS
Shaker
MLS
Vortex-Genie 2T Mixer
VWR 14216-188, VWR 14216-186
Vortex Adapter-60, for use with Vortex-Genie
AM10014
Consumables
Disposable gloves
MLS
Micropipette tips, aerosol-resistant
MLS
Pipettors:
MLS
• Positive-displacement
• Air-displacement
• Multichannel
Pipettes
MLS
Tubes, conical, 50-mL
AM12502
Microcentrifuge tubes, non-stick RNase-free,
1.5-mL, 1 box (250 tubes/box)
AM12450
Safe-Lock PCR clean microcentrifuge tubes,
round-bottom, 2-mL, 1 bag (100 tubes/bag)
VWR 62111-754
Reagents
Ethanol, 95%
MLS
IMPORTANT! Do not use denatured ethanol
because it contains components not compatible
with the protocol.
Isopropanol, 100%
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
MLS
3
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Pharmaceutical Testing
Materials not included
in the kit
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Required materials
Equipment, consumables, and reagents (continued)
Item
DNase-free, sterile-filtered water
4
Source
MLS
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Preparation and extraction of lysate DNA
Overview
For the isolation of DNA from difficult-to-dissolve, protein-rich tissue samples or
samples containing Gram-positive bacteria, Applied Biosystems recommends the
Proteinase K lysis protocol described on page 8. For all other samples, use the
chemical lysis protocol described on page 7. After sample lysis using one of these
two protocols, proceed with the DNA extraction protocol described on page 11.
For the isolation and purification of DNA from broth culture samples,
Applied Biosystems recommends that the volume of the liquid sample vary from
20 to 100 µL. For liquid samples with a volume >100 µL, centrifuge the liquid
sample at 16000 × g for 5 minutes in an Eppendorf microcentrifuge (to pellet
microorganisms), then discard the supernatant. The recommended elution volume
is 50 µL. This volume may be increased to 200 µL as necessary.
Before you begin
Before starting your sample extraction:
• Prepare the following reagents:
– Binding Solution – Refer to the procedure that is described on page 2.
– Wash Buffer – Refer to the procedure that is described on page 2.
• Power on the refrigerated centrifuge to allow it to cool down before use.
• If only one block heater is available, set the temperature to 56 °C, then reset
to 70 °C after the 56 °C incubation so that the heater is at the required
temperature when needed. If two block heaters are available, set one to 56 °C
and the other to 70 °C.
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
5
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Pharmaceutical Testing
Preparation and extraction of lysate DNA
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Preparation and extraction of lysate DNA
Sample processing
workflow
The figure below shows a sample processing workflow. The Proteinase K lysis
protocol is recommended for difficult-to-dissolve, protein-rich tissue samples or
samples containing Gram-positive bacteria. The chemical lysis protocol is
recommended for all other samples.
Chemical lysis protocol
Proteinase K lysis protocol
Step 1: Place the sample in a 1.5 mL-tube.
Step 1: Place the sample in a 1.5 mL-tube.
Step 2: Add 300 µL of Lysis Buffer. Vortex for 15 sec.
Step 2: Add 200 µL of Proteinase K (PK) Buffer. Mix
and resuspend.
Step 3: Incubate at 70 °C for 20 min.
Step 3: Add 10 µL of Proteinase K (20 mg/mL).
Step 4: Vortex for 10 sec at maximum speed.
Step 5a: Vortex the magnetic particles for 5 sec.
Bind DNA
Step 5b: Add 30 µL of Magnetic Particles to the sample
mix and vortex for 10 sec at low speed.
Step 4: Incubate at 56 °C for 20 min.
Step 5: Add 400 µL of Lysis Buffer. Vortex for 15 sec.
Note: If necessary you can leave the lysate at room
temperature for up to two hours.
Step 6: Vortex the sample lysate for 10 sec at maximum
speed.
Step 6: Add 190 µL of Binding Solution. Vortex for 5 sec.
Step 7a: Vortex the magnetic particles for 5 sec.
Step 7: Incubate with shaking for 7 min.
Step 7b: Add 30 µL of Magnetic Particles to the sample
mix and vortex for 10 sec at low speed.
Step 8a: Vortex for 10 sec at low speed.
Step 8b: Place the tube in the magnetic stand.
Step 8c: Aspirate the supernatant.
Bind DNA
Step 8: Add 400 µL of Binding Solution and vortex for 5 sec.
Step 9: Incubate with shaking for 10 min at room temp.
Step 10a – 10c: Vortex for 10 sec at low speed. Place the
tube in the magnetic stand and aspirate.
;
Pellet of magnetic particles with bound DNA;
Proceed to DNA extraction (page 9)
Figure 1
6
Sample processing using the PrepSEQ Nucleic Acid Extraction Kit
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Preparation and extraction of lysate DNA
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Pharmaceutical Testing
Chemical lysis protocol
For the following hazards, see the complete safety alert descriptions in
Appendix A on page 29:
DANGER! CHEMICAL HAZARD. Lysis Buffer.
WARNING! CHEMICAL HAZARD. Magnetic Particles.
1. Place the sample in a 1.5 mL microcentrifuge tube.
2. Add 300 µL of the Lysis Buffer to the tube, then vortex the tube for 15
seconds, until the sample is completely resuspended.
3. Incubate the tube at 70 °C for 20 minutes.
4. Vortex the tube for 10 seconds at maximum speed.
5. Add the Magnetic Particles to the sample mix:
a. Vortex the stock Magnetic Particles for approximately 5 seconds, until
resuspension is complete.
b. Add 30 µL of the Magnetic Particles to the sample mix, then vortex the
tube for 10 seconds at low speed.
6. Add 190 µL of the Binding Solution to the tube, then vortex the tube for 5
seconds.
7. Incubate the mix with shaking (approximately 1000 rpm) at room
temperature for 7 minutes.
8. Separate the mix:
a. Vortex the tube for 10 seconds at low speed.
b. Place tube in the magnetic stand.
Note: Complete separation of the magnetic particles pellet containing
the bound DNA occurs in 1 to 2 minutes.
c. Aspirate the supernatant carefully, then discard as much of the liquid as
possible without disturbing the magnetic particles pellet.
9. Proceed to “DNA extraction: purification of DNA” on page 11.
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
7
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Preparation and extraction of lysate DNA
Proteinase K lysis
protocol
For the following hazards, see the complete safety alert descriptions in
Appendix A on page 29:
DANGER! CHEMICAL HAZARD. Lysis Buffer.
WARNING! CHEMICAL HAZARD. Proteinase K Buffer.
WARNING! CHEMICAL HAZARD. Proteinase K.
WARNING! CHEMICAL HAZARD. Magnetic Particles.
IMPORTANT! Use proper aseptic technique while handling samples to avoid
cross-contamination.
1. Place the sample in a 1.5-mL microcentrifuge tube.
2. Add 200 µL of the Proteinase K (PK) Buffer. Mix and resuspend.
3. Add 10 µL of Proteinase K (20 mg/mL) to the sample.
4. Incubate the tube at 56 °C for 20 minutes.
5. Add 400 µL of the Lysis Buffer, then vortex the tube for 15 seconds until the
sample is completely resuspended.
Note: If necessary you can leave the lysate at room temperature for up to 2
hours.
6. Vortex the sample lysate for 10 seconds at maximum speed.
7. Add the Magnetic Particles to the sample mix:
a. Vortex the Magnetic Particles for approximately 5 seconds, until
resuspension is complete.
b. Add 30 µL of the Magnetic Particles to the sample mix, then vortex the
tube for 10 seconds at low speed.
Note: If a white precipitate forms in the magnetic particles tube, refer to
the IMPORTANT paragraph on page 2.
8. Add 400 µL of the Binding Solution to the tube, then vortex the tube for 5
seconds.
9. Incubate with shaking (approximately 1000 rpm) for 10 minutes at room
temperature.
8
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Preparation and extraction of lysate DNA
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Pharmaceutical Testing
10. Separate the mix:
a. Vortex the tube for 10 seconds at low speed.
b. Place the tube in the magnetic stand with the magnetic particles pellet
oriented toward the magnet.
Note: Complete separation of the magnetic particles containing the
bound DNA occurs in 1 to 2 minutes.
c. Carefully aspirate, then discard as much of the supernatant as possible
without disturbing the magnetic particles pellet.
11. Proceed to “DNA extraction: purification of DNA” on page 11.
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
9
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Preparation and extraction of lysate DNA
DNA extraction
workflow
The figure below shows a DNA extraction workflow. For details, go to page 11.
Wash DNA
Step 1: Add 300 µL of Wash Solution.
Step 2a– 2c: Vortex for 10 sec at setting #7. Place the tubes into a magnetic
stand. Aspirate the supernatant without disturbing the magnetic particles pellet.
Step 3: Repeat steps 1 and 2 two more times.
Step 4: Leave the tube lid open for 5 min to air-dry.
Elute DNA
Step 1 Add 50 µL of Elution Buffer.
Step 2: Vortex for 5 sec at medium speed.
Step 3: Incubate at 70 °C for 5 min.
Step 4: Vortex for 2 sec at medium speed.
Step 5: Place the tube into a magnetic stand.
Step 6: Transfer the liquid phase to a non-stick tube.
Step 7: Store at –20 °C.
Figure 2
10
DNA extraction using the PrepSEQ Nucleic Acid Extraction Kit
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Preparation and extraction of lysate DNA
Wash DNA
1. Add 300 µL of Wash Solution to the tube.
2. Separate the mix:
a. Vortex the tube for 10 seconds at speed setting #7 until the pellet is
completely resuspended.
b. Place the tube in the magnetic stand with the magnetic particles pellet
oriented toward the magnet.
Note: Complete capture of the magnetic particles containing the bound
DNA occurs in 30 seconds.
c. Carefully aspirate, then discard as much of the supernatant as possible
without disturbing the magnetic particles pellet. The pellet contains
bound DNA.
3. Repeat step 1 and step 2 two more times.
4. With the tube lid open, air-dry the magnetic particles pellet in the magnetic
stand for 5 minutes at room temperature.
IMPORTANT! Ethanol in the Wash Solution decreases recovery and causes
PCR inhibition. Avoid carrying over ethanol into the elution steps.
Elute DNA
For the following hazard, see the complete safety alert descriptions in Appendix A
on page 25:
WARNING! CHEMICAL HAZARD. Elution Buffer.
1. Add 50 µL of Elution Buffer to the tube.
Note: The magnetic particles may be difficult to detach from the tube wall.
Place the tube in the benchtop microcentrifuge with the magnetic particles
pellet oriented toward the center, then spin the tube for 30 seconds to detach
the magnetic particles into the Elution Buffer.
2. Vortex the tube for 5 seconds at medium speed.
3. Incubate the tube at 70 °C for 5 minutes.
4. Vortex the tube for 2 seconds at medium speed.
5. Place the tube in the magnetic stand for at least 2 minutes (or until magnetic
particles pellet is formed).
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
11
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Pharmaceutical Testing
DNA extraction:
purification of DNA
Chapter 1 PrepSEQ™ Nucleic Acid Extraction Kit for Pharmaceutical Testing
Troubleshooting
6. Transfer the liquid phase containing the eluted DNA to a non-stick 1.5-mL
microcentrifuge tube.
Note: The final liquid phase contains extracted DNA.
7. Store the tube at –20 °C.
Troubleshooting
Observation
Possible Cause
Action
Poor extraction efficiency
(low yields)
• Too much starting material
• Spin speed is too high (pellet too
hard) in the preparation step
• Start with less volume (less material)
• Pellet using a slower speed
• Ensure resuspension of the pellet in Lysis Buffer or
Proteinase K Buffer before proceeding
Carryover of ethanol into Elute DNA
step 1 on page 11.
Thoroughly air-dry the magnetic particles pellet in the
magnetic stand (no longer than 15 minutes) at room
temperature.
Magnetic particles are attached too
tightly to the tube wall during
Elution DNA step 1 on page 11.
Spin the tube for 30 seconds at maximum speed to
detach the magnetic particles into the Elution Buffer.
Magnetic particles are difficult to
resuspend during Elute DNA step 1
on page 11.
Incubate the pellets at 70 °C for 7 minutes. Vortex the
tubes 3 times during incubation to facilitate
resuspension.
12
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 2
PrepSEQ™ Nucleic Acid Extraction Kit
for Food Testing
Product overview
Required materials
Kit contents
The PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing (PN 4400799)
contains reagents for 100 reactions. Kit components are shown in the table below.
For information on the kit contents, refer to the “Materials supplied” section in the
packaging insert for your kit.
Item
Quantity or
volume
Lysis Buffer, 2 bottles
50 mL/bottle
Magnetic Particles, 2 tubes
1.5 mL/tube
Binding Solution (Isopropanol), 1 empty bottle
NA
Wash Buffer Concentrate, 2 bottles
26 mL/bottle
Elution Buffer, 1 bottle
25 mL
Proteinase K (PK) Buffer, 1 bottle
50 mL
Proteinase K, 1 tube
1.25 mL
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
13
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Food Testing
The PrepSEQ™ Nucleic Acid Extraction Kit prepares high quality microbial DNA
and RNA from broth cultures.
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Required materials
Storage
For the following hazards, see the complete safety alert descriptions in
Appendix A on page 29:
DANGER! CHEMICAL HAZARD. Lysis Buffer.
WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate.
WARNING! CHEMICAL HAZARD. Elution Buffer.
WARNING! CHEMICAL HAZARD. Proteinase K Buffer.
WARNING! CHEMICAL HAZARD. Proteinase K.
WARNING! CHEMICAL HAZARD. Magnetic Particles.
• Room temperature:
– Lysis Buffer
– Binding Solution – Before its use for the first-time, add 30 mL of 100%
isopropanol to the empty Binding Solution bottle. Label the bottle to
indicate that isopropanol is added.
– Wash Buffer – Before its use for the first-time, add 74 mL of 95%
ethanol to the Wash Buffer Concentrate bottle, mix well, then label the
bottle to indicate that ethanol is added.
– Elution Buffer
– Proteinase K Buffer
• Magnetic Particles: 2 to 8 °C
IMPORTANT! White precipitate occasionally forms in the magnetic particles
tube. Extraction experiments show that formation of precipitate does not
affect performance. If precipitate forms, incubate the tube at 37 °C for 10
minutes, then vortex to completely resuspend the particles.
• Proteinase K: –20 °C
For information on storage of kit components, refer to the “Storage” section in the
packaging insert for your kit.
14
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Required materials
Materials not included
in the kit
The following table includes materials for using (but not included in) the
PrepSEQ™ Nucleic Acid Extraction Kit. Unless otherwise indicated, many of the
listed items are available from major laboratory suppliers (MLS).
Equipment, consumables, and reagents
Item
Source
Equipment
MLS
MagMAX™ Express-96 Deep Well Magnetic
Particle Processor
PN 4400079
Stomacher 400 Laboratory Blender
Seward #0400/001/AJ
Vortexer
MLS
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Food Testing
Benchtop microcentrifuge (Eppendorf 5415 D or
equivalent)
Consumables
Disposable gloves
MLS
Micropipette tips, aerosol-resistant
MLS
Pipettors:
MLS
• Positive-displacement
• Air-displacement
• Multichannel
MagMAX™ Express-96 Deep Well Tip Combs
PN 4388487
MagMAX™ Express-96 Deep Well Plates
PN 4388476
Microtiter 96-Well Plate
VWR #11388-570,
Thermofisher #95040410
Whirl-Pak Filter Bags, 6” × 9”, 24 oz, 250/pkg
(Stomacher bags with mesh)
VWR #11216-520
Whirl-Pak Filter Bags, 6” × 9”, 24 oz (Stomacher
bags without mesh)
VWR #11216-280
Microcentrifuge tubes, PCR clean, 1.5-mL
MLS
Reagents
Bacterial growth media ‡
MLS
DNase-free, sterile-filtered water
PN AM9938
Ethanol, 95%
MLS
Isopropanol, 100%
MLS
‡ microorganism appropriate
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
15
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Preparation and extraction of lysate DNA
Preparation and extraction of lysate DNA
Overview
The PrepSEQ™ Nucleic Acid Extraction Kit is designed to work with most food
types. The kit protocol involves:
• Sample enrichment
• Sample extraction
• Lysis
To isolate DNA from Gram-negative bacteria in food samples, the chemical lysis
protocol that is described on page 19 is recommended.
To isolate DNA from Gram-positive bacteria in food samples, the Proteinase K
lysis protocol that is described on page 21 is recommended.
For the isolation and purification of DNA from overnight broth culture samples, a
1-mL sample volume is recommended. The recommended elution volume is
140 µL.
If you prepare food samples that yield large pellet samples, such as that found
with chocolate, the PrepSEQ™ preclarification protocol on page 19 is
recommended.
Before you begin
Before starting your sample extraction:
• Prepare the following reagents:
– Binding Solution – See the procedure that is described on page 14.
– Wash Buffer – See the procedure that is described on page 14.
• Vortex the Magnetic Particles, then keep them at room temperature.
16
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Preparation and extraction of lysate DNA
Kit workflow
The figure below shows a sample processing workflow based on the chemical
lysis protocol. For details go to page 19.
Enrichment
Use bacterial growth media appropriate for microorganisms and enrich according to
your standard protocols.
Chemical lysis protocol
Step 1: Transfer 1 mL of sample into a 1.5-mL tube.
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Food Testing
Step 2: Centrifuge the tube for 3 min at 16000 × g. Discard the supernatant.
If your sample produces a large food pellet
(PrepSEQ™ preclarification protocol):
– Transfer 1 mL of sample into a 1.5-mL tube.
– Centrifuge the tube for 1 min at 4000 × g.
– Collect the supernatant, then transfer to a
new 1.5-mL tube.
Step 3: Add 300 µL of Lysis Buffer, then resuspend.
Step 4: Transfer the sample to a microtiter 96-well deep well plate.
Step 5a–b: Prepare the plates.
Step 6: Select 44000799DWPrepSEQGN from the MagMAX™ Express magnetic
particle processor, then press Start.
Step 7a–e: Load the plates, then press Start.
Step 8: After 18 min, dispense the Binding Mix:
a. Vortex the Magnetic Particles for 5 sec until resuspension is complete.
b. Add 30 µL of Magnetic Particles. Swirl the plate.
c. Add 180 µL of Binding Solution. Swirl the plate.
d. Load the plate into the instrument, then press Start.
Step 9: After 30 min, sample preparation is complete. The message “Enjoy your DNA”
is displayed on the screen. Remove the elution plate.
Proceed with PCR, or seal the plate and store at –20 °C.
Figure 1 Food sample preparation using the PrepSEQ™ Nucleic Acid
Extraction Kit with the chemical lysis protocol
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
17
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Preparation and extraction of lysate DNA
Kit workflow
The figure below shows a sample processing workflow based on the Proteinase K
lysis protocol. For details go to page 21.
Enrichment
Use bacterial growth media appropriate for microorganisms and enrich according to
your standard protocols.
Proteinase K lysis protocol
Step 1: Transfer 1 mL into a 1.5-mL tube.
Step 2: Centrifuge the tube for 3 min at 16000 × g. Discard the supernatant.
Step 3: Add 200 µL of PK Buffer. Mix and resuspend.
Step 4: Add 10 µL of Proteinase K (20 mg/mL).
Step 5: Transfer the sample to a microtiter 96-well deep well plate.
Step 6a–b: Prepare the plates.
Step 7: Select 44000799DWPrepSEQGP from the MagMAX™ Express magnetic
particle processor, then press Start.
Step 8a–e: Load plates, then press Start.
Step 9: After 20 min, dispense the Lysis Buffer:
a. Add 300 µL of Lysis Buffer. Swirl the plate.
b. Load the plate into the instrument, then press Start.
Step 10: After 18 min, dispense the Binding Mix:
a. Vortex Magnetic Particles for 5 sec.
b. Add 30 µL of Magnetic Particles to each well. Swirl the plate.
c. Add 300 µL of Binding Solution to each well. Swirl the plate.
d. Load the plate into the instrument, then press Start.
Step 11: After 30 min, sample preparation is done. The message “Enjoy your DNA”
is displayed on the screen. Remove the elution plate.
Proceed with PCR, or seal the plate and store at –20 °C.
Figure 2 Food sample preparation using the PrepSEQ™ Nucleic Acid
Extraction Kit with the Proteinase K lysis protocol
18
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Preparation and extraction of lysate DNA
Generic enrichment
Getting started
• For isolation of DNA from Gram-positive bacteria, proceed to the “Chemical
lysis protocol” below.
• For isolation of DNA from Gram-negative bacteria, proceed to the
“Proteinase K lysis protocol” on page 21.
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Food Testing
Chemical lysis protocol
Use bacterial growth media appropriate for microorganisms and enrich according
to your standard protocols.
For the following hazards, see the complete safety alert descriptions in
Appendix A on page 29:
DANGER! CHEMICAL HAZARD. Lysis Buffer.
WARNING! CHEMICAL HAZARD. Elution Buffer.
WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate.
WARNING! CHEMICAL HAZARD. Magnetic Particles.
IMPORTANT! Use proper aseptic technique while handling samples to avoid
cross-contamination.
1. Transfer 1 mL of sample into a 1.5-mL microcentrifuge tube.
2. Centrifuge the tube for 3 minutes at 16000 × g.
Note: Discard the supernatant without disturbing the pellet. Remove liquid
as quickly as possible to prevent dissipation of pellet.
If your sample produces a large pellet at this step, such as found with
chocolate food samples, the following PrepSEQ™ preclarification protocol is
recommended:
• Transfer 1 mL of sample into a 1.5-mL microcentrifuge tube.
• Centrifuge the tube containing your sample for 1 minute at 4000 × g.
• Transfer supernatant to a new 1.5-mL microcentrifuge tube, without
disturbing the pellet.
• Proceed to step 3.
3. Add 300 µL of the Lysis Buffer to the tube. Resuspend by pipetting up and
down, or vortex until the pellet is resuspended. Quick spin for 5 seconds to
remove the Lysis Buffer from the tube lid.
4. Transfer the sample to a sterile microtiter 96-well deep well (DW) plate
(PN 4388476).
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
19
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Preparation and extraction of lysate DNA
5. Prepare the plates:
a. To prepare the elution plate, add 140 µL of Elution Buffer to those wells
of the microtiter 96-well plate that correspond to the microtiter 96-well
DW plate containing sample.
b. To prepare wash plates 1 and 2, add 300 µL of Wash Buffer to those
wells of the microtiter 96-well DW plate by that correspond to the
microtiter 96-well DW plate containing sample.
6. Select 44000799DWPrepSEQGN from the MagMAX™ Express magnetic
particle processor. Press Start.
7. Load the plates according to the readout. Verify orientation {A1 to A1}.
a. Tip combs – in microtiter 96-well plate; press Start.
b. Elution plate (140 µL of Elution Buffer) – In microtiter 96-well plate;
press Start.
c. Wash plate 2 (300 µL of Wash Buffer) – In microtiter 96-well DW plate;
press Start.
d. Wash plate 1 (300 µL of Wash Buffer) – In microtiter 96-well DW plate;
press Start.
e. Lysis plate (sample in Lysis Buffer) – In microtiter 96-well DW plate;
press Start.
8. After 18 minutes, the MagMAX™ Express magnetic particle processor
prompts you to dispense the Binding Mix.
a. Vortex the Magnetic Particles for 5 seconds until resuspension is
complete.
b. Add 30 µL of the Magnetic Particles to each well. Swirl the plate.
c. Add 180 µL of Binding Solution to each well. Swirl the plate.
d. Load the plate back into the instrument. Press Start.
9. When sample preparation is complete, the message “Enjoy your DNA” is
displayed on the screen. Remove the elution plate. Store at –20 °C.
IMPORTANT! For PCR, add 30 µL of eluate to the lyophilized assay.
20
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Preparation and extraction of lysate DNA
Proteinase K lysis
protocol
For the following hazards, see the complete safety alert descriptions in
Appendix A on page 29:
DANGER! CHEMICAL HAZARD. Lysis Buffer.
WARNING! CHEMICAL HAZARD. Proteinase K Buffer.
WARNING! CHEMICAL HAZARD. Proteinase K.
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Food Testing
WARNING! CHEMICAL HAZARD. Elution Buffer.
WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate.
WARNING! CHEMICAL HAZARD. Magnetic Particles.
IMPORTANT! Use proper aseptic technique while handling samples to avoid
cross-contamination.
Note: Premix Proteinase K Buffer and Proteinase K prior to dispensing.
1. Transfer 1 mL of sample into a 1.5-mL microcentrifuge tube.
2. Centrifuge the tube for 3 minutes at 16000 × g.
Note: Discard the supernatant without disturbing the pellet. Remove liquid
as quickly as possible to prevent dissipation of pellet.
3. Add 200 µL of the Proteinase K (PK) Buffer. Mix, then resuspend.
Note: Depending on the number of samples to be run, a master mix may be
prepared prior to dispensing; master mix volume = N × (200 µL of
Proteinase K Buffer + 10 µL of Proteinase K) where N = # of samples.
4. Add 10 µL of Proteinase K (20 mg/mL) to the sample.
5. Transfer the sample to a sterile microtiter 96-well deep well (DW) plate
(PN 4388476).
6. Prepare the plates:
a. To prepare the elution plate, add 140 µL of Elution Buffer to those wells
of the microtiter 96-well plate that correspond to the microtiter 96-well
DW plate containing sample.
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
21
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Preparation and extraction of lysate DNA
b. To prepare wash plates 1 and 2, add 300 µL of Wash Buffer to those
wells of the microtiter 96-well DW plate by that correspond to the
microtiter 96-well DW plate containing sample.
7. Select 44000799DWPrepSEQGP program from the MagMAX™ Express
magnetic particle processor. Press Start.
8. Load the plates according to the readout. Verify orientation {A1 to A1}.
a. Tip combs – in microtiter 96-well plate; press Start.
b. Elution plate (140 µL Elution Buffer) – In microtiter 96-well plate;
press Start.
c. Wash plate 2 (300 µL of Wash Buffer) – In microtiter 96-well DW plate;
press Start.
d. Wash plate 1 (300 µL of Wash Buffer) – In microtiter 96-well DW plate;
press Start.
e. Lysis plate (sample in PK Buffer) – In microtiter 96-well DW plate;
press Start.
9. After 20 minutes, the MagMAX™ Express magnetic particle processor
prompts you to dispense the Lysis Buffer.
a. Add 300 µL of Lysis Buffer to each well.
b. Load the plate into the instrument. Press Start.
10. After 18 minutes, the instrument prompts you to dispense the Binding Mix.
a. Vortex the stock Magnetic Particles for 5 seconds until resuspension is
complete.
b. Add 30 µL of Magnetic Particles to each well. Swirl the plate.
c. Add 300 µL of Binding Solution to each well. Swirl the plate.
d. Load the plate into the instrument. Press Start.
11. When sample preparation is complete, the message “Enjoy your DNA” is
displayed on the screen. Remove the elution plate. Store at –20 °C.
IMPORTANT! For PCR, add 30 µL of eluate to the lyophilized assay.
22
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Troubleshooting
Troubleshooting
For food testing
Observation
Inhibition of PCR,
indicated by nondetection of IPC reaction
Possible cause
Magnetic particles were in the
elution plate
Action
Avoid disturbing the magnetic particles during
transfer of eluted DNA to the lyophilized assay.
Optional:
Or:
Place the elution plate on the 96-well magnetic ring
stand (PN AM10050) during transfer of sample to the
lyophilized assay.
Elution plate contains incompletely
removed particulate residue from
food sample
Avoid residue during transfer of eluted DNA to
lyophilized assay.
Optional:
Spin the plate for 30 seconds at 4000 × g to pellet the
food residue to the bottom of the plate.
The bacterial pellet is
hard to avoid during
removal of supernatant
The bacterial pellet is
difficult to resuspend
Removal of sample supernatant
before addition of lysis buffer was
incomplete
Ensure maximal removal of the supernatant without
disturbing the bacterial pellet.
The sample was left unattended
before aspirating off the
supernatant, causing dissipation of
the bacterial pellet
Remove the supernatant immediately following
centrifugation.
The size of the bacterial pellet is
very small and difficult to see
Remove the supernatant carefully, leaving behind up
to 50 µL of supernatant, to avoid aspiration of pellet.
Pellet is too hard
Ensure maximum resuspension of the pellet in the
Lysis Buffer or Proteinase K Buffer before
proceeding.
Transfer the entire contents, including the
incompletely resuspended pellet (if any) to the Lysis
Plate.
The pellet is too large
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Apply PrepSEQ™ preclarification protocol.
23
PrepSEQ™ Nucleic Acid Extraction Kit
Protocol for Food Testing
Spin the plate for 30 seconds at 4000 × g to pellet the
magnetic particles to the bottom of the plate.
Chapter 2 PrepSEQ™ Nucleic Acid Extraction Kit for Food Testing
Troubleshooting
24
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Appendix A
Safety
This appendix covers:
■ Chemical safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
■ Chemical waste safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
■ Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
■ Chemical alerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
25
Appendix A Safety
Chemical safety
Chemical safety
Chemical hazard
warning
WARNING! CHEMICAL HAZARD. Before handling any chemicals,
refer to the Material Safety Data Sheet (MSDS) provided by the
manufacturer, and observe all relevant precautions.
WARNING! CHEMICAL HAZARD. All chemicals in the instrument,
including liquid in the lines, are potentially hazardous. Always determine
what chemicals have been used in the instrument before changing reagents
or instrument components. Wear appropriate eyewear, protective clothing,
and gloves when working on the instrument.
WARNING! CHEMICAL HAZARD. Four-liter reagent and waste bottles
can crack and leak. Each 4-liter bottle should be secured in a low-density
polyethylene safety container with the cover fastened and the handles
locked in the upright position. Wear appropriate eyewear, clothing, and
gloves when handling reagent and waste bottles.
WARNING! CHEMICAL STORAGE HAZARD. Never collect or store
waste in a glass container because of the risk of breaking or shattering.
Reagent and waste bottles can crack and leak. Each waste bottle should be
secured in a low-density polyethylene safety container with the cover
fastened and the handles locked in the upright position. Wear appropriate
eyewear, clothing, and gloves when handling reagent and waste bottles.
Chemical safety
guidelines
26
To minimize the hazards of chemicals:
• Read and understand the Material Safety Data Sheets (MSDSs) provided by
the chemical manufacturer before you store, handle, or work with any
chemicals or hazardous materials. (See “About MSDSs” on page 27.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers
open. Use only with adequate ventilation (for example, fume hood). For
additional safety guidelines, consult the MSDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow
the manufacturer’s cleanup procedures as recommended in the MSDS.
• Comply with all local, state/provincial, or national laws and regulations
related to chemical storage, handling, and disposal.
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Appendix A Safety
Chemical waste safety
About MSDSs
Chemical manufacturers supply current Material Safety Data Sheets (MSDSs)
with shipments of hazardous chemicals to new customers. They also provide
MSDSs with the first shipment of a hazardous chemical to a customer after an
MSDS has been updated. MSDSs provide the safety information you need to
store, handle, transport, and dispose of the chemicals safely.
Each time you receive a new MSDS packaged with a hazardous chemical, be sure
to replace the appropriate MSDS in your files.
Obtaining
MSDSs
The MSDS for any chemical supplied by Applied Biosystems is available to you
free 24 hours a day. To obtain MSDSs:
1. Go to www.appliedbiosystems.com, click Support, then select MSDS.
2. In the Keyword Search field, enter the chemical name, product name, MSDS
part number, or other information that appears in the MSDS of interest.
Select the language of your choice, then click Search.
3. Find the document of interest, right-click the document title, then select any
of the following:
• Open – To view the document
• Print Target – To print the document
• Save Target As – To download a PDF version of the document to a
destination that you select
Note: For the MSDSs of chemicals not distributed by Applied Biosystems,
contact the chemical manufacturer.
Chemical waste safety
Chemical waste
hazards
CAUTION! HAZARDOUS WASTE. Refer to Material Safety Data
Sheets (MSDSs) and local regulations for handling and disposal.
WARNING! CHEMICAL WASTE HAZARD. Wastes produced by
Applied Biosystems instruments are potentially hazardous and can cause
injury, illness, or death.
WARNING! CHEMICAL STORAGE HAZARD. Never collect or store
waste in a glass container because of the risk of breaking or shattering.
Reagent and waste bottles can crack and leak. Each waste bottle should be
secured in a low-density polyethylene safety container with the cover
fastened and the handles locked in the upright position. Wear appropriate
eyewear, clothing, and gloves when handling reagent and waste bottles.
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
27
Appendix A Safety
Chemical waste safety
Chemical waste safety
guidelines
To minimize the hazards of chemical waste:
Waste disposal
If potentially hazardous waste is generated when you operate the instrument, you
must:
• Read and understand the Material Safety Data Sheets (MSDSs) provided by
the manufacturers of the chemicals in the waste container before you store,
handle, or dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary waste container
holds the immediate waste. A secondary container contains spills or leaks
from the primary container. Both containers must be compatible with the
waste material and meet federal, state, and local requirements for container
storage.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers
open. Use only with adequate ventilation (for example, fume hood). For
additional safety guidelines, consult the MSDS.
• Handle chemical wastes in a fume hood.
• After emptying a waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in accordance with
good laboratory practices and local, state/provincial, or national
environmental and health regulations.
• Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and
disposed of according to all local, state/provincial, and/or national
regulations.
IMPORTANT! Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.
28
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Appendix A Safety
Biological hazard safety
Biological hazard safety
General biohazard
WARNING! BIOHAZARD. Biological samples such as tissues, body
fluids, infectious agents, and blood of humans and other animals have the
potential to transmit infectious diseases. Follow all applicable local,
state/provincial, and/or national regulations. Wear appropriate protective
equipment, which includes but is not limited to: protective eyewear, face
shield, clothing/lab coat, and gloves. All work should be conducted in
properly equipped facilities using the appropriate safety equipment (for
example, physical containment devices). Individuals should be trained
according to applicable regulatory and company/institution requirements
before working with potentially infectious materials. Read and follow the
applicable guidelines and/or regulatory requirements in the following:
– U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories (stock no.
017-040-00547-4; bmbl.od.nih.gov)
– Occupational Safety and Health Standards, Bloodborne Pathogens
(29 CFR§1910.1030; www.access.gpo.gov/
nara/cfr/waisidx_01/29cfr1910a_01.html)
– Your company’s/institution’s Biosafety Program protocols for working
with/handling potentially infectious materials.
Additional information about biohazard guidelines is available at:
www.cdc.gov
Chemical alerts
General alerts for all
chemicals
Specific chemical alerts
Avoid contact with (skin, eyes, and/or clothing). Read the MSDS, and follow the
handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
WARNING! Dispose of the pipette tips and other contaminated materials
in biohazard waste.
DANGER! CHEMICAL HAZARD. Lysis Buffer contains guanidine
thiocyanate. Exposure causes eye burns and skin irritation. It is harmful if
inhaled, absorbed through the skin or if swallowed. Contact with acids or
bleach liberates toxic gases. DO NOT ADD acids or bleach to any liquid
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
29
Appendix A Safety
Chemical alerts
wastes containing DNA Binding Buffer. Avoid breathing vapor. Do not
taste or swallow. Use with adequate ventilation. Read the MSDS, and
follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves.
WARNING! CHEMICAL HAZARD. Wash Buffer Concentrate
contains a material that may cause eye, skin, and respiratory tract irritation,
and adverse effects on the kidneys and blood and central nervous system.
Use with adequate ventilation. Read the MSDS, and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and gloves.
WARNING! CHEMICAL HAZARD. Elution Buffer contains a material
that may cause eye, skin, and respiratory tract irritation, and adverse effects
on the kidneys and blood and central nervous system. Read the MSDS, and
follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves.
WARNING! CHEMICAL HAZARD. Proteinase K Buffer contains a
material that may cause eye, skin, and respiratory tract irritation, and
adverse effects on the kidneys and blood and central nervous system. Read
the MSDS, and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
WARNING! CHEMICAL HAZARD. Proteinase K causes eye, skin, and
respiratory tract irritation. Exposure may cause an allergic reaction. Read
the MSDS, and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
WARNING! CHEMICAL HAZARD. Magnetic Particles cause eye,
skin, and respiratory tract irritation. Exposure may cause an allergic
reaction. Harmful by inhalation, skin absorption and if swallowed. Do not
taste or swallow. Avoid breathing vapor (or dust). Use with adequate
ventilation. Avoid contact with eyes and skin. Read the MSDS, and follow
the handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.
30
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Documentation
Documentation
Related documentation
For additional documentation, see “How to obtain support” on page vi.
For information on new assays and updated product documentation, go to
http://info.appliedbiosystems.com/pathogenkits
Document title
PrepSEQ™
Nucleic Acid Extraction Kit Quick Reference Card
™
PN
4406303
PrepSEQ Nucleic Acid Extraction Kit Protocol: Salmonella spp.
4405968
PrepSEQ™
4405967
Nucleic Acid Extraction Kit Quick Reference Card: Salmonella
spp.
PrepSEQ™ Nucleic Acid Extraction Kit Protocol: Listeria monocytogenes
4405966
PrepSEQ™
Nucleic Acid Extraction Kit Quick Reference Card: Listeria
monocytogenes
4405965
PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit Protocol
4401253
PrepSEQ™
Mycoplasma Nucleic Acid Extraction Kit Quick Reference Card
4406304
PrepSEQ™
Rapid Spin Sample Preparation Kit Protocol
4412847
™
PrepSEQ Rapid Spin Sample Preparation Kit Quick Reference Card
4412846
PrepSEQ™
4412848
Rapid Spin Sample Preparation Kit Protocol: Salmonella spp.
PrepSEQ™
Rapid Spin Sample Preparation Kit Quick Reference Card:
Salmonella spp.
4412849
PrepSEQ™ Rapid Spin Sample Preparation Kit Protocol: Listeria
monocytogenes
4412851
PrepSEQ™ Rapid Spin Sample Preparation Kit Quick Reference Card:
Listeria monocytogenes
4412852
MicroSEQ® Listeria monocytogenes Detection Kit Protocol
4405962
®
MicroSEQ Listeria monocytogenes Detection Kit Quick Reference Card
4405961
MicroSEQ®
4405964
Salmonella spp. Detection Kit Protocol
®
4405963
®
MicroSEQ Mycoplasma Real-Time PCR Detection Kit Protocol
4393111
MicroSEQ®
4393471
MicroSEQ Salmonella spp. Detection Kit Quick Reference Card
Mycoplasma Real-Time PCR Detection Kit Quick Reference
Card
Portable document format (PDF) versions of this guide and the documents listed
above are available at www.appliedbiosystems.com
Note: To open the documentation available from the Applied Biosystems web
site, use the Adobe® Acrobat® Reader® software available at www.adobe.com
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
31
Documentation
Related documentation
32
PrepSEQ™ Nucleic Acid Extraction Kit Protocol
Part Number 4400739 Rev. C
06/2010
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