QUICK REFERENCE
PrepSEQ® Sample Preparation Kits
Publication Part Number 4465875 Rev. A Revision Date August 2011
Note: For safety and biohazard guidelines, refer to the “Safety” section in the PrepSEQ® Sample Preparation Kits User Guide
(Part No. 4465957). For every chemical, read the SDS and follow the handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.
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Choose a sample preparation protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
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Prepare reagents and instruments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
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PrepSEQ® 1-2-3 protocol for Mycoplasma or MMV detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
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PrepSEQ® large-scale protocol for Mycoplasma detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
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PrepSEQ® 3-in-1 protocol for Mycoplasma, MMV, and Vesivirus detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
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Kit contents and storage conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Choose a sample preparation protocol
There are three sample lysis protocols for use with the PrepSEQ® Sample Preparation Kits. Use Table 1 to select the appropriate protocol
and kit based on your sample type and detection task.
Table 1 Select a sample preparation protocol
Protocol
Purpose
For use with kit
PrepSEQ® 1-2-3 protocol for Mycoplasma
or MMV detection
To process 100 µL (up to 106 cells) sample volume
for detection of Mycoplasma and/or MMV†
PrepSEQ® 1-2-3 Nucleic Acid
Extraction Kit
PrepSEQ® large-scale protocol for
Mycoplasma detection
To process sample volume to up 50 mL (up to
2 × 108 cells) for detection of Mycoplasma
PrepSEQ® Mycoplasma Nucleic
Acid Extraction Kit
PrepSEQ® 3-in-1 protocol for
Mycoplasma, MMV, and Vesivirus detection
To process 100 µL (up to 106 cells) sample volume
for detection of Mycoplasma, MMV, and Vesivirus†
PrepSEQ® 1-2-3 Nucleic Acid
Extraction Kit
† For samples with greater than 106 total cells: Centrifuge the sample at 500 × g for 2 minutes, then use 100 µL of the supernatant.
Prepare reagents and instruments
Before beginning the sample preparation protocol:
1. Review “Required Materials” and “Sample Preparation Guidelines” in the PrepSEQ® Sample Preparation Kits User Guide
(Part No. 4465957).
2. Prepare the following reagents before their first-time use:
• Binding Solution – Add 30 mL of 100% isopropanol to the empty Binding Solution bottle. Mark the bottle label to indicate that
isopropanol has been added.
• Wash Buffer – Add 74 mL of 95% ethanol to the Wash Buffer Concentrate bottle, mix well, then mark the bottle label to indicate
that ethanol has been added.
3. Incubate the Magnetic Particles at 37°C for 10 minutes, then vortex the Magnetic Particles at medium speed until the particles are
completely resuspended.
Note: White precipitate occasionally forms in the Magnetic Particles tubes. It does not affect performance, but may make them difficult
to pipet. Always incubate the particles for at 37°C for 10 minutes before use.
Note: During extraction, when you place tubes into the Magnetic Stand, always orient the magnetic
particles pellet toward the magnet.
4. If you are performing the large-scale protocol:
• Place aliquots of 1✕ PBS on ice. You need 300 µL PBS per sample. When not in use, store 1✕ PBS
at 2–8°C.
• Power on the refrigerated centrifuge to allow it to cool down before use.
PrepSEQ® Sample Preparation Kits
5. Power on the heat blocks:
• 1-2-3 protocol or large-scale protocol – Heat block settings for required incubation: 37°C, 56°C, and 70°C.
• 3-in-1 protocol – Heat block settings for required incubation: 37°C and 45°C.
PrepSEQ® 1-2-3 protocol for Mycoplasma or MMV detection
1
Prepare samples
Place the following in a new lock-safe 2-mL microcentrifuge tube:
• For samples with up to 106 total cells – Use 100 µL of sample
• For samples with greater than 106 total cells – Centrifuge the sample at 500 × g for
2 minutes, then use 100 µL of the supernatant.
2
Prepare sample lysate
For each sample tube:
a. Add 200 µL of Lysis Buffer, then vortex for approximately 5 seconds to mix.
b. Add 2 µL of 0.5M EDTA and 18 µL RNase Cocktail, then briefly vortex to mix.
c. Incubate at 56°C for 15 minutes.
d. Add 2 µL of Proteinase K, then briefly vortex to mix.
e. Incubate at 56°C for 10 minutes.
f. Incubate at room temperature for 5 minutes.
g. Add 700 µL of Lysis Solution. Vortex for approximately 5 seconds to mix.
3
Bind DNA
For each tube of sample lysate:
a. Add 30 µL of Magnetic Particles, then vortex.
b. Add 525 µL of Binding Solution, then invert the tube to mix.
c. Vortex at medium speed for 5 minutes, using a vortex adaptor, to capture the nucleic acid.
d. Centrifuge in a microcentrifuge by pressing the short spin button for 15 seconds, then releasing
the button. During this time, the microcentrifuge should reach top speed.
e. Place in the Magnetic Stand for 5 minutes.
f. Aspirate the supernatant without disturbing the magnetic particles, then discard the
supernatant.
4
Wash DNA
For each tube of magnetic particles pellet (bound DNA):
a. Add 300 µL of Wash Buffer.
b. Vortex for approximately 5 seconds.
c. Centrifuge in a microcentrifuge by pressing the short spin button for 15 seconds, then releasing
the button. During this time, the microcentrifuge should reach top speed.
d. Place in the Magnetic Stand for one minute.
e. Aspirate the supernatant without disturbing the magnetic particles, then discard the
supernatant.
f. Repeat steps 1 through 5.
g. Use a P200 pipette to aspirate the residual supernatant from the bottom of the tube, then discard
the supernatant.
h. With the lid open, air-dry the magnetic particles pellet at room temperature for 5 minutes to
remove any remaining ethanol.
5
Elute DNA
For each sample:
a. Add 100 µL of Elution Buffer.
b. Vortex for approximately 10 seconds.
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PrepSEQ® Sample Preparation Kits
c. Incubate at 70°C for 7 minutes. Vortex 2 to 3 times during incubation to ensure complete
resuspension of the magnetic particles.
d. Centrifuge at top speed for 5 minutes.
e. Place in the Magnetic Stand for three minutes.
f. Transfer the eluate to a non-stick 1.5-mL microcentrifuge tube.
The extracted DNA is now ready for use in the appropriate PCR assay, or it may be stored at –20°C
if not used immediately.
3
PrepSEQ® Sample Preparation Kits
PrepSEQ® large-scale protocol for Mycoplasma detection
IMPORTANT! For the large-scale protocol, you have three options for preparing the sample lysate:
• Option 1: Process cell culture media only
• Option 2: Process cell culture media and mammalian cells separately
• Option 3: Process cell culture media and mammalian cells pooled together
1
Separate mammalian
cells from cell culture
media
For all options:
a. Place the cell culture sample (up to ~ 2 × 108 total cells) in a conical tube.
b. Centrifuge the tube at 1,000 × g for 5 minutes to pellet the mammalian cells.
c. Transfer the supernatant to a new conical tube, and keep on ice. The supernatant contains free
Mycoplasma.
d. If you are performing:
• Option 1 – Discard the mammalian cell pellet.
• Option 2 or 3 – Place the mammalian cell pellet on ice.
2
Process the supernatant
(cell culture media)
For all options:
a. Centrifuge the conical tube with the supernatant at 16,000 × g for 30 minutes to pellet the
Mycoplasma.
b. Aspirate and discard the supernatant without disturbing the Mycoplasma pellet.
c. If you are performing:
• Option 1 – Add 300 µL of PBS, then pipet up and down to resuspend the Mycoplasma pellet.
Transfer the resuspended pellet to a 2-mL microcentrifuge tube. Proceed to step 4 on page 4.
• Option 2 – Add 300 µL of PBS, then pipet up and down to resuspend the Mycoplasma pellet.
Transfer the resuspended pellet to a 2-mL microcentrifuge tube. Keep the resuspended
Mycoplasma pellet on ice while you process the mammalian cell pellet.
• Option 3 – Keep the Mycoplasma pellet on ice.
3
(Options 2 and 3 only)
Process the mammalian
cell pellet
For options 2 and 3 only:
a. Add 550 µL of ice-cold Cell Fractionation Buffer to the mammalian cell pellet. Very gently pipet
up and down several times with a P1000 to completely resuspend the mammalian cells.
b. Transfer the mammalian cell suspension to a 2-mL microcentrifuge tube, then incubate on ice
for 5 minutes.
c. Centrifuge the 2-mL tube at 1000 × g for 10 minutes at 4°C to pellet the cellular membranes and
nuclei.
d. If you are performing:
• Option 2 – Use a P200 pipette to transfer 300 µL (two 150-µL aliquots) of the cell
fractionation supernatant (mammalian cell lysate) to a new 2-mL microcentrifuge tube,
without disturbing the viscous cellular material. Keep the tube on ice.
• Option 3 – Use a P200 pipette to transfer 300 µL (two 150-µL aliquots) of the cell
fractionation supernatant (mammalian cell lysate) to the Mycoplasma pellet tube from step 3
in "Process the supernatant (cell culture media)" above without disturbing the viscous
cellular material, then pipette up and down to resuspend the Mycoplasma pellet. Transfer the
pooled cell culture media and mammalian cell lysate to a new 2-mL microcentrifuge tube.
4
Treat samples with
RNase cocktail and
Proteinase K
For all options:
Note: If you are performing option 2, Separately process the resuspended Mycoplasma (from the
cell culture media) and the cell fractionation supernatant (from the mammalian cell pellet).
a. Add 2 µL of 0.5 M EDTA and 18 µL of RNase Cocktail, then briefly vortex the 2-mL tube to mix.
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PrepSEQ® Sample Preparation Kits
b. Incubate the tube at 56°C for 30 minutes to digest the cellular RNA. Vortex twice during
incubation.
c. Add 5 µL of Proteinase K, then briefly vortex to mix.
d. Incubate at 56°C for 10 minutes.
e. Add 700 µL of Lysis Buffer, then vortex to mix well.
5
Bind DNA
For all options, for each tube of sample lysate:
a. Add 30 µL of Magnetic Particles, then vortex.
b. Add 525 µL of Binding Solution, then invert the tube to mix.
c. Vortex at medium speed for 5 minutes, using a vortex adaptor, to capture the nucleic acid.
d. Centrifuge in a microcentrifuge by pressing the short spin button for 15 seconds, then releasing
the button. During this time, the microcentrifuge should reach top speed.
e. Place in the Magnetic Stand for 5 minutes.
f. Aspirate the supernatant without disturbing the magnetic particles, then discard the
supernatant.
6
Wash DNA
For all options, for each tube of magnetic particles pellet (bound DNA):
a. Add 300 µL of Wash Buffer.
b. Vortex for approximately 5 seconds.
c. Centrifuge in a microcentrifuge by pressing the short spin button for 15 seconds, then releasing
the button. During this time, the microcentrifuge should reach top speed.
d. Place in the Magnetic Stand for one minute.
e. Aspirate the supernatant without disturbing the magnetic particles, then discard the
supernatant.
f. Repeat steps 1 through 5.
g. Use a P200 pipette to aspirate the residual supernatant from the bottom of the tube, then discard
the supernatant.
h. With the lid open, air-dry the magnetic particles pellet at room temperature for 5 minutes to
remove any remaining ethanol.
7
Elute DNA
For all options, for each sample:
a. Add 100 µL of Elution Buffer.
b. Vortex for approximately 10 seconds.
c. Incubate at 70°C for 7 minutes. Vortex 2 to 3 times during incubation to ensure complete
resuspension of the magnetic particles.
d. Centrifuge at top speed for 5 minutes.
e. Place in the Magnetic Stand for three minutes.
f. Transfer the eluate to a non-stick 1.5-mL microcentrifuge tube.
The extracted DNA is now ready for use in the appropriate PCR assay, or it may be stored at –20°C
if not used immediately.
5
PrepSEQ® Sample Preparation Kits
PrepSEQ® 3-in-1 protocol for Mycoplasma, MMV, and Vesivirus detection
1
Prepare samples
Place the following in a new lock-safe 2-mL microcentrifuge tube:
• For samples with up to 106 total cells – Use 100 µL of sample
• For samples with greater than 106 total cells – Centrifuge the sample at 500 × g for
2 minutes, then use 100 µL of the supernatant.
2
Prepare sample lysate
For each sample tube:
a. Add 500 µL of Lysis Buffer, then vortex for approximately 15 seconds to mix.
b. Incubate at 45°C for 10 minutes.
c. Vortex approximately 10 seconds to mix.
3
Bind nucleic acid
For each sample lysate tube:
a. Add 30 µL of Magnetic Particles, then vortex.
b. Add 330 µL of Binding Solution, then invert the tube to mix.
c. Vortex at medium speed for 10 minutes, using a vortex adaptor, to capture the nucleic acid.
d. Centrifuge in a microcentrifuge by pressing the short spin button for 15 seconds, then releasing
the button. During this time, the microcentrifuge should reach top speed.
e. Place in the Magnetic Stand for 5 minutes.
f. Aspirate the supernatant without disturbing the magnetic particles, then discard the
supernatant.
4
Wash nucleic acid
For each tube of magnetic particles pellet (bound DNA):
a. Add 300 µL of Wash Buffer.
b. Vortex for approximately 5 seconds.
c. Centrifuge in a microcentrifuge by pressing the short spin button for 15 seconds, then releasing
the button. During this time, the microcentrifuge should reach top speed.
d. Place in the Magnetic Stand for one minute.
e. Aspirate the supernatant without disturbing the magnetic particles, then discard the
supernatant.
f. Repeat steps 1 through 5.
g. Use a P200 pipette to aspirate the residual supernatant from the bottom of the tube, then discard
the supernatant.
h. With the lid open, air-dry the magnetic particles pellet at room temperature for 5 minutes to
remove any remaining ethanol.
5
Elute nucleic acid
For each sample:
a. Add 100 µL of Elution Buffer.
b. Vortex for approximately 10 seconds.
c. Incubate at 45°C for 5 minutes. Vortex 2 to 3 times during incubation to ensure complete
resuspension of the magnetic particles.
d. Centrifuge at top speed for 5 minutes.
e. Place in the Magnetic Stand for three minutes.
f. Transfer the eluate to a non-stick 1.5-mL microcentrifuge tube.
The extracted DNA is now ready for use in the appropriate PCR assay, or it may be stored at –20°C
if not used immediately.
6
PrepSEQ® Sample Preparation Kits
Kit contents and storage conditions
• The PrepSEQ® 1-2-3 Nucleic Acid Extraction Kit can be ordered as a stand-alone kit.
• The PrepSEQ® Mycoplasma Nucleic Acid Extraction Kit must be ordered as part of the one of the following Mycoplasma detection kits:
– MycoSEQ™ Mycoplasma Detection Assay with Discriminatory Positive Control and PrepSEQ® Mycoplasma Sample Preparation Kits
User Guide and Quick Reference included. (Part No. 4460627)
– MycoSEQ™ Mycoplasma Detection Assay with Discriminatory Positive Control and PrepSEQ® Mycoplasma Sample Preparation Kits
User Guide and Quick Reference not included (Part No. 4460626)
Kit components may be shipped separately depending on configuration and storage conditions. Use the lists in Table 2 and Table 3 to
confirm that you have received all components.
Table 2 PrepSEQ® 1-2-3 Nucleic Acid Extraction Kit (Part No. 4452222) contains reagents for 100 small-scale (100 µL) cell culture
extractions
Shipped in box labeled
Component
Description
Part number†
Storage
RNase Cocktail
RNase Cocktail
One 1.0-mL tube
AM2286
– 20°C
Box 1
Lysis Buffer
Two 50-mL bottles
4400659
Room temp.
Binding Solution (Isopropanol)
One empty bottle
4400789
Wash Buffer Concentrate
Two 26-mL bottles
4400783
Elution Buffer
One 25-mL bottle
4400784
Proteinase K (PK) Buffer
One 50-mL bottle
4400787
Box 2
Magnetic Particles
Two 1.5-mL tubes
4401405
2–8°C
Box 3
Proteinase K (20 mg/mL)
One 1.25-mL tube
4403958
–20°C
† These part numbers are provided for identification purposes; the items cannot be ordered separately.
Table 3 PrepSEQ® Mycoplasma Nucleic Acid Extraction Kit contains reagents for 100 small-scale (100 to 2000 µL) or 100 large-scale
(2 to 10 mL) cell culture extractions
Shipped in box labeled
Description
Cell Fractionation Buffer
Three 25-mL bottles
RNase Cocktail
Box 1
Description
Part number†
4405889
Storage
2–8°C
Two 1.0-mL tubes
4405890
– 20°C
Lysis Buffer
Two 50-mL bottles
4400659
Room temp.
Binding Solution (Isopropanol)
One empty bottle
4400789
Wash Buffer Concentrate
Two 26-mL bottles
4400783
Elution Buffer
One 25-mL bottle
4400784
Proteinase K (PK) Buffer
One 50-mL bottle
4400787
Box 2
Magnetic Particles
Two 1.5-mL tubes
4401405
2–8°C
Box 3
Proteinase K (20 mg/mL)
One 1.25-mL tube
4403958
–20°C
† These part numbers are provided for identification purposes; the items cannot be ordered separately.
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PrepSEQ® Sample Preparation Kits
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
NOTICE TO PURCHASER: PLEASE REFER TO THE PREPSEQ MYCOPLASMA NUCLEIC ACID EXTRACTION KIT AND PREPSEQ 1-2-3 NUCLEIC ACID EXTRACTION KIT PRODUCT INSERT AND
PROTOCOL FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION.
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