PrepSEQ™ Residual DNA Sample Preparation
Kit
USER GUIDE
Automated and manual protocols for preparation of samples for
use with resDNASEQ™ Quantitative DNA Kits
Catalog Number 4413686
Publication Number 4469838
Revision F
For Research Use Only. Not for use in diagnostic procedures.
Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom
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The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. 4469838
Revision Date Description
F
E 12 Dec 2019 Added the resDNASEQ™ Quantitative HEK293 DNA Kit
5 August 2020
Added the resDNASEQ™ Quantitative Sf9 and Baculovirus DNA Kit
(Cat. No. A46066).
(Cat. No. A46014).
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 5
Product description ............................................................. 5
Contents and storage ............................................................ 5
Required materials not supplied for manual protocols ................................ 6
Required materials not supplied for automated protocols ............................. 7
Workflow ....................................................................... 9
■
CHAPTER 2 Prepare the reagents and samples ................................ 10
Prepare the reagents: before first use of the kit .................................... 10
Magnetic beads ........................................................... 10
Binding Solution ........................................................... 10
Wash Buer Concentrate ................................................... 10
Prepare reagents: before each use of the kit ....................................... 10
Proteinase K (PK) mix ...................................................... 10
Lysis solution ............................................................. 11
Guidelines for optimal DNA yields ................................................ 11
Sample preparation guidelines ................................................... 12
Sample dilution (if necessary) ............................................... 12
Triplicate extractions ....................................................... 12
Extraction control guidelines ................................................ 13
■
■
PrepSEQ
CHAPTER 3 Manual protocol for residual DNA extraction ..................... 14
Digest the test samples and controls ............................................. 14
Bind the DNA .................................................................. 14
Wash the DNA ................................................................. 15
Elute the DNA ................................................................. 16
CHAPTER 4 Automated protocol for residual DNA extraction ................ 17
Before each use of the kit ....................................................... 17
Ensure that you have the correct plates ....................................... 17
Prepare the plates ......................................................... 17
Prepare the lysis plate .......................................................... 18
Process samples on the instrument .............................................. 18
™
Residual DNA Sample Preparation Kit User Guide
3
Contents
■
■
■
■
APPENDIX A Troubleshooting .................................................... 20
APPENDIX B Good laboratory practices ....................................... 21
Good laboratory practices for PCR and RT-PCR ................................... 21
Avoiding false positives due to cross-contamination ........................... 21
Plate layout suggestions ....................................................... 21
APPENDIX C Safety ............................................................... 22
Chemical safety ................................................................ 23
Biological hazard safety ......................................................... 24
Documentation and support ....................................................... 25
Related documentation ......................................................... 25
Customer and technical support ................................................. 25
Limited product warranty ........................................................ 26
4
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
1
IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
Product description
The PrepSEQ™ Residual DNA Sample Preparation Kit extracts residual DNA from
products that are produced in cell lines such as CHO, E. coli, HEK293, Human,
MDCK, NS0, Pichia, Sf9 and Baculovirus, and Vero. The kit uses chemical lysis
and magnetic beads to extract genomic DNA from diverse sample types, including
samples that contain high protein and low DNA concentration.
For quantification of host-cell line residual DNA, we recommend using the
resDNASEQ™ Quantitative DNA Kits as described in the resDNASEQ™ Quantitative
DNA Kits User Guide (Pub. No. 4469836). To ensure accurate quantitative results,
extract each sample in triplicate and perform a single PCR reaction for each
extraction.
Product information
Contents and storage
The kit contains reagents sucient for 100 extractions.
Table 1 PrepSEQ™ Residual DNA Sample Preparation Kit (Cat. No. 4413686)
Contents
Box 1, PrepSEQ™ Nucleic Acid Extraction Kit
Lysis Buer 2 × 50 mL
Binding Solution (Isopropanol), empty bottle 1
Wash Buer Concentrate 2 × 26 mL
Elution Buer 25 mL
Proteinase K (PK) Buer
Can be used for existing validated manual
protocols.
Amount Storage
50 mL
Room temperature
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
5
Chapter 1 Product information
1
Required materials not supplied for manual protocols
Table 1 PrepSEQ Residual DNA Sample Preparation Kit (Cat. No. 4413686) (continued)
Contents Amount Storage
Proteinase K (PK) Buer II
Recommended for new manual protocols.
Required for automated protocols.
Box 2, PrepSEQ™ Nucleic Acid Extraction Kit
Magnetic Particles 2 × 1.5 mL Room temperature
Box 3, PrepSEQ™ Nucleic Acid Extraction Kit
Proteinase K, 20 mg/mL 1.25 mL –20°C or below
PrepSEQ™ Residual DNA Sample Preparation Kit
Proteinase K, 20 mg/mL 1.25 mL
Yeast tRNA, 10 mg/mL 0.5 mL
Glycogen, 5 mg/mL 2 × 1.0 mL
[1]
Also sold separately (Cat. No. 4415320).
[1]
Room temperature50 mL
–20°C or below
Required materials not supplied for manual protocols
Unless otherwise indicated, all materials are available through thermofisher.com.
MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.
Item
Equipment
Pharma Magnetic Stand-96 or
Magnetic stand, 16-position
Block heater for use with 2‑mL tubes.
Manual DNA extraction involves two incubations at
dierent settings, so two heaters may be convenient.
Benchtop microcentrifuge for 1.5‑mL and 2‑mL tubes MLS
Vortex-Genie™ 2T Mixer VWR™ 14216-188, VWR™ 14216-186
Vortex Adapter-60, for use with the Vortex-Genie
Consumables
Disposable gloves MLS
Aerosol-resistant micropipette tips MLS
™
Major laboratory supplier (MLS)
Source
A31543
12321D
AM10014
6
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Chapter 1
Required materials not supplied for automated protocols
Item Source
Product information
1
PIPETMAN™ Pipettors, P1000, P200, P20 and P10:
•
Positive-displacement
•
Air-displacement
•
Multichannel
Nonstick, RNaseZap™-free Microfuge Tubes, 1.5‑mL (1
box; 250 tubes/box)
Safe-Lock PCR clean microcentrifuge tubes, roundbottom, 2‑mL
Reagents
Ethanol, 95%
MLS
AM12450
VWR™ 62111-754
MLS
IMPORTANT! Do not use denatured ethanol. It
contains components that are not compatible with the
protocol.
Isopropanol, 100% MLS
5 M NaCl and 1 N NaOH solutions MLS
Hydrochloric acid (HCI) MLS
PBS solution, 1X, pH 7.4, free of Mg and Ca (if needed to
dilute samples before DNA extraction)
14190094
Required materials not supplied for automated protocols
Unless otherwise indicated, all materials are available through thermofisher.com.
MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.
Table 2 Pharma KingFisher™ Flex-96 Deep Well Magnetic Particle Processor
(Cat. No. A31508) and Pharma MagMAX™ Express-96 instrument
Item
Pharma MagMAX™ Express-96 DW plate A31540
Pharma MagMAX™ Express-96 Deep-Well Tip
Combs
Pharma KingFisher™ Flex Magnetic Head for 96
Deep-Well Plate
Pharma MagMAX™ 96 PCR Well Magnetic Head 4472991
Pharma MagMAX™ Express-96 Standard Plates A31541
[1]
This instrument is no longer available for purchase
Source
A31537
A31542
[1]
accessories
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
7
Chapter 1 Product information
1
Required materials not supplied for automated protocols
Table 3 Additional materials
Consumables
Disposable gloves MLS
Aerosol-resistant micropipette tips MLS
Item Source
PIPETMAN™ Pipettors, P1000, P200, P20 and P10:
•
Positive-displacement
•
Air-displacement
•
Multichannel
Nonstick, RNaseZap™-free Microfuge Tubes, 1.5‑mL
(1 box; 250 tubes/box)
Reagents
Ethanol, 95%
MLS
AM12450
MLS
IMPORTANT! Do not use denatured ethanol. It
contains components that are not compatible with
the protocol.
Isopropanol, 100% MLS
5 M NaCl and 1 N NaOH solutions MLS
Hydrochloric acid (HCI) MLS
PBS solution, 1X, pH 7.4, free of Mg and Ca (if
needed to dilute samples before DNA extraction)
14190094
8
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Workflow
Chapter 1 Product information
Workflow
1
Manual residual DNA extraction
Digest the test samples and controls
(page 14)
▼ ▼
Bind the DNA (page 14) Prepare the lysis plate (page 18)
▼ ▼
Wash the DNA (page 15)
▼
Elute the DNA (page 16)
Automated residual DNA extraction
Prepare the plates (page 17)
Process samples on the instrument
(page 18)
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
9
Prepare the reagents and samples
2
Prepare the reagents: before first use of the kit
Magnetic beads
1.
Set a block heater to 37°C.
2.
Incubate the Magnetic Particle suspension at 37°C for a minimum of 10 minutes
with intermittent vortexing at setting #7, or until the particles are completely
suspended.
Binding Solution
1.
Add 30 mL of 100% isopropanol to the Binding Solution bottle.
2.
Label the bottle to indicate that it contains isopropanol, then store the bottle at
ambient temperature.
Wash Buer Concentrate
1.
Add 74 mL of 95% ethanol to one bottle of PrepSEQ™ Wash Buer Concentrate,
then mix completely.
2.
Label the bottle to indicate that it contains ethanol, then store the bottle at room
temperature.
Prepare reagents: before each use of the kit
Proteinase K (PK) mix
•
Use Proteinase K (PK) Buer II for new manual protocols and automated
protocols.
Note: Proteinase K (PK) Buer is also provided in the kit for use with existing
manual protocols that have been internally validated with this buer.
•
Prepare a fresh mix before each use of the kit.
•
Include a 10% overage to account for pipetting losses.
10
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Chapter 2
Number of extractions
Component
1 7 10 13 25
Proteinase K, 20 mg/mL 10 μL 70 μL 100 μL 130 μL 250 μL
Prepare the reagents and samples
Guidelines for optimal DNA yields
2
Proteinase K (PK) Buer II or
Proteinase K (PK) Buer
60 μL 420 μL 600 μL 780 μL 1,500 μL
Lysis solution
•
Prepare a fresh mixture immediately before use or during Proteinase K
incubation.
•
Prepare 360 µL (amount required) of lysis solution mix per sample.
Reagent
Glycogen, 5 mg/mL 180 µL
Yeast tRNA, 10 mg/mL 4 µL
Lysis Buer 7,600 µL
Total 7,784 µL
Guidelines for optimal DNA yields
•
Maintain a homogenous suspension of the magnetic beads to maximize the
surface area to which the DNA can bind. The appearance of the mixture should
be homogenous after mixing.
•
After drying, the DNA remains bound to the magnetic beads. Do not allow the
magnetic beads to over-dry because this reduces the elution eciency; overdried beads are not easily resuspended.
•
During manual elution, vortex every 2 minutes to assist elution. This will result in
better yield during recovery.
Volume for ~20 extractions
Note: Some test samples cause the beads to adhere very firmly to the tube wall,
while others form loose pellets that detach during the vortex steps. All pellets
should dissolve with vortexing during heated elution. If vortexing does not result
in full resuspension, then wash the beads o the tube by pipetting.
Note: White or brown precipitate may form in the Magnetic Particles tube if it
is stored at 2–8°C. The precipitate will dissolve when it is heated to 37°C for a
minimum of 10 minutes with intermittent vortexing. Make sure the precipitate is
completely dissolved before using the beads.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
11
Chapter 2 Prepare the reagents and samples
2
Sample preparation guidelines
Sample preparation guidelines
Sample dilution (if necessary)
Test samples from the early purification process often contain levels of DNA that are
above the highest point of the residual DNA assay standard curve. You must dilute
these samples (from 1:100 up to 1:10,000) before PrepSEQ™ Residual DNA sample
preparation.
•
Dilute test samples before DNA extraction with a solution of 1X PBS (pH 7.4; free
of Mg and Ca) or 50 mM Tris, pH 8.0, 0.5 M NaCl.
Note: Diluting samples in water or TE reduces extraction eciency.
•
Use the sample dilution buer as the negative extraction control instead of water.
•
Alternatively, dilute extracted DNA with elution buer before running the PCR
reaction.
Triplicate extractions
Triplicate extractions are required for post-PCR analysis calculation of mean quantity,
standard deviation, and coecient of variation.
In addition to test samples, we recommend triplicate extractions for controls (for an
explanation of controls , see “Extraction control guidelines” on page 13).
Perform a single PCR reaction for each extraction.
The table below illustrates the total number of extractions required based on the 1, 2,
and 3 samples extracted in a batch.
Table 4 Total number of extractions per batch of test samples
Number of test samples
1 3 extractions required for each:
2 15
3 21
[1]
Optional during routine testing.
•
•
•
Test sample
Test sample
extraction/recovery control
(ERC)
Negative extraction control
Total number of extractions for
the batch
9
[1]
12
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Chapter 2 Prepare the reagents and samples
Sample preparation guidelines
Extraction control guidelines
We recommend that you use the following extraction controls:
Type of control Contains Number to run Used to
2
Negative (NEG)
Extraction/recovery (ERC) Positive control from the
[1]
Optional during routine testing.
[1]
1X PBS 1 per batch of
resDNASEQ™ Quantitative
DNA Kit
For the Extraction/recovery (ERC) :
•
Prepare the control standard dilutions as described in resDNASEQ™ Quantitative
DNA Kits User Guide (Pub. No. 4469836).
•
Add a volume of positive control standard dilution to each test sample that yields
a PCR input DNA amount that is 2–10 times the amount of DNA measured in the
test sample without the addition of the DNA control.
For example:
–
The DNA amount measured in a test sample is ≤1 pg.
–
To prepare a 10 pg ERC for a PCR elution volume of 50 µL, spike samples
with 16.7 µL of the 3 pg/µL positive control standard dilution (SD3) = 50 pg
spike to yield 10 pg per PCR reaction.
•
Prepare three separate extractions for each test sample, then add the ERC to
each reaction. Do not prepare a large volume of ERC, then aliquot it into three
reactions.
extractions
3 per sample
Test for cross-contamination of DNA
extraction reagents.
•
Evaluate the eciency of
DNA extraction, recovery,
and quantification from test
samples.
•
Verify reagent and system
performance.
Note: To calculate the eciency of DNA recovery and quantification from the test
samples, subtract the amount of DNA measured in the sample without the addition of
DNA control from the amount of DNA measured in the ERC sample.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
13
Manual protocol for residual DNA
3
Digest the test samples and controls
1.
Set a block heater to 56°C. If available, set a second block heater to 70°C.
2.
Label 2‑mL Safe-Lock tubes:
•
3 for each sample
•
3 for each sample + ERC
•
3 for NEG
3.
Add 100 µL of sample, sample + ERC, or 1X PBS to into each tube.
extraction
Bind the DNA
4.
Add 10 µL of 5 M NaCl and 70 μLProteinase K/Proteinase K Buer II mix.
5.
Briefly vortex and centrifuge.
6.
Incubate at 56°C for 30 minutes.
If only one block heater is available, after this incubation step is complete, reset
the block heater to 70°C for the elution step.
Note: For samples with high protein concentration, extending the incubation
time to 60 minutes can increase recovery.
7.
Cool samples to room temperature.
8.
Add 360 µL freshly made Lysis solution mix to each tube.
1.
Vortex the Magnetic Particles to resuspend the particles.
Note: The appearance of the mixture should be homogeneous.
2.
Add 30 µL of the Magnetic Particles to each tube.
14
3.
Add 400 µL Binding Solution to each tube.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Wash the DNA
Chapter 3
4.
Mix and vortex the tubes:
a.
Close the caps, immediately invert each tube twice to mix.
b.
Vortex the tubes in the vortex adaptor for 5 minutes at setting #7.
5.
Briefly centrifuge the tubes for 15 seconds at top speed (>15,000 × g) to collect
the Magnetic Particles at the bottom of the tubes.
6.
Place the tubes in the magnetic stand with the pellet against the magnet, then
let the tubes stand for 5 minutes or until the solution is clear.
7.
Without disturbing the magnetic beads, remove the supernatant using a
PIPETMAN™ pipette or by aspiration.
For aspiration of liquid supernatants and wash buers during sample preparation, we
recommend attaching the waste-collection bottle to the vacuum using tubing that
can accommodate 200‑µL pipette tips.
Manual protocol for residual DNA extraction
Wash the DNA
3
1.
Remove the tube rack (with tubes) from the magnetic stand, then add 300 µL of
Wash Solution to the tubes. Vortex the tubes for 5 seconds at room temperature
at setting #7.
2.
Centrifuge the tubes in a microcentrifuge at top speed (>15,000 × g) for a
maximum of 20 seconds. Do not centrifuge for >20 seconds.
3.
Place the tubes in the magnetic stand, then let the tubes stand for 1 minute.
Note: The Magnetic Particles with the bound DNA are magnetically captured
after approximately 1 minute.
4.
Without disturbing the Magnetic Particles, remove the supernatant using a
PIPETMAN™ pipette or by aspiration.
5.
Remove the tube rack (with tubes) from the magnetic stand, then add 300 µL of
Wash Solution to each tube for a second wash. Vortex the tubes for 5 seconds
at room temperature at setting #7.
6.
Centrifuge the tubes in a microcentrifuge at top speed (>15,000 × g) for a
maximum of 20 seconds. Do not centrifuge for >20 seconds.
7.
Place the tubes in the magnetic stand, then let the tubes stand for 1 minute.
Note: The Magnetic Particles with the bound DNA are magnetically captured
after approximately 1 minute.
8.
Open all tubes, then start the 5‑minute timer.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
15
3
Chapter 3
Elute the DNA
Manual protocol for residual DNA extraction
Elute the DNA
9.
Without disturbing the Magnetic Particles, remove the supernatant using a
PIPETMAN™ pipette or by aspiration.
Use a P200 to remove the remaining solution from the bottom of the tube.
10.
With the tube lid open, air-dry the Magnetic Particles pellet in the magnetic
stand for no more than 5 minutes at room temperature.
IMPORTANT! Air-dry to remove ethanol from the Wash Solution. After dried,
the DNA stays bound to the magnetic beads. Do not over-dry; over-dried beads
are not easily resuspended.
1.
Add 50 µL of Elution Buer to each tube.
2.
Vortex the tubes for 20 seconds at high speed, then incubate the tubes at 70°C
for 7 minutes. Vortex the tubes two to three times during the incubation to help
resuspension.
Note: (Optional) If vortexing does not result in full resuspension, then wash the
beads o the tube by pipetting.
3.
Centrifuge the tubes in a microcentrifuge at top speed (>15,000 × g) for a
maximum of 20 seconds. Do not centrifuge for >20 seconds.
4.
Place the tubes in the magnetic stand, then let the tubes stand for 1 minute.
Note: The Magnetic Particles with the bound DNA are magnetically captured
after approximately 1 minute.
5.
Without disturbing the Magnetic Particles, transfer the liquid phase containing
the eluted DNA to a new nonstick 1.5‑mL microcentrifuge tube.
6.
Centrifuge the tube at top speed (>15,000 × g) for 3 minutes to collect the
Magnetic Particles at the bottom, then place the tubes in the magnetic stand for
1 minute.
7.
Without disturbing the Magnetic Particles, transfer the liquid phase containing
the eluted DNA to the nonstick 1.5‑mL microcentrifuge tube.
Note: Magnetic Particles can inhibit PCR.
When you finish the sample extraction procedure, see the resDNASEQ™ Quantitative
DNA Kits User Guide (Pub. No. 4469836) to set up PCR for DNA quantification.
16
Use 10 µL of the eluate in the real-time PCR.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Automated protocol for residual
4
You can use the KingFisher™ Flex or MagMAX™ Express 96-deep well automation
platforms to automate the extraction of host-cell line residual DNA. For all chemicals,
read the Safety Data Sheet (SDS) and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
Before each use of the kit
Ensure that you have the correct plates
The KingFisher™ Flex or the MagMAX™ Express require 5 plates.
DNA extraction
Prepare the plates
Plate name
Lysis 96 deep-well plate
Wash 1 96 deep-well plate
Wash 2 96 deep-well plate
Elution 96 deep-well plate
Comb loading plate 96 deep-well tip comb combined with 96
Prepare the Wash 1, Wash 2, and Elution plates:
Plate name
Wash 1 96 deep-well plate 300 µL of Wash buer
Wash 2 96 deep-well plate 300 µL of Wash buer
Elution 96 deep-well plate 200 µL of Elution buer
Plate type Volume of buer to add
Plate type
standard plate
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
17
Chapter 4
4
Prepare the lysis plate
Automated protocol for residual DNA extraction
Prepare the lysis plate
In all steps that require pipetting, dispense liquid at bottom center of the wells.
1.
Add 100 μL to the appropriate wells of the 96 deep-well Lysis plate:
•
3 wells for each sample
•
3 wells for each sample + ERC
•
3 wells for NEG
2.
Add 10 μL of 5 M NaCl to each sample well.
3.
Add 70 µL Proteinase K/Proteinase K (PK) Buer II mix to each sample well.
Process samples on the instrument
1.
Select the script or program for the instrument you are using:
Instrument
KingFisher™ Flex PrepSEQ_resDNA_v1 script
MagMAX™ Express-96 PrepSEQ_resDNA_2011
PrepSEQ_1hr_resDNA (if installed)
2.
Load the plates into the instrument in the order listed below. After loading each
plate, press START to move the turntable.
a.
Comb loading plate
b.
Elution plate with 200 μL of Elution Buer
c.
Wash 2 plate with 300 μL of wash buer
d.
Wash 1 plate with 300 μL of wash buer
e.
Lysis plate
3.
Press START to begin the PK digestion process.
The instrument mixes the samples for 10 seconds at fast speed, then incubates
the samples at 56°C for 30 minutes, mixing at slow speed. When digestion
is complete, the instrument pauses and returns the Lysis plate to the loading
position.
Select
18
4.
After the digestion step is complete, add additional components to the Lysis
plate:
a.
Remove the Lysis plate from the instrument.
b.
Add 360 µL of Lysis Solution to each sample well.
c.
Add 30 µL of Magnetic Particle suspension to each sample well.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Chapter 4 Automated protocol for residual DNA extraction
Process samples on the instrument
d.
Add 400 µL of Binding Solution to each sample well, then immediately pipet
up-and-down three times to mix.
e.
Place the plate back into the instrument loading position, then press START
to begin binding.
5.
When DNA extraction is finished, the instrument returns the Elution plate to the
loading position.
4
When you finish the sample extraction procedure, refer to the resDNASEQ
Quantitative DNA Kits User Guide (Pub. No. 4469836) to set up PCR for DNA
quantitation.
Note: Use 10 µL of the eluate in the real-time PCR.
™
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
19
A
Troubleshooting
Observation
Poor extraction eciency (low yields) Overdrying the sample. Start the 5‑minute timer before
Magnetic Particles are dicult to
resuspend during the elution.
Formation of precipitate in Magnetic
Particles.
PK Buer was used instead of PKII
Buer.
Possible cause Action
removing ~300 µL from the first 6–
8 samples. Then continue removing
wash buer from the remaining
samples.
Incubate the pellets at 70°C for
>7 minutes. Vigorously vortex the
tubes three times during incubation
to help resuspension.
Do not overdry.
If necessary, repeat the incubation
and vortexing steps.
Incubate the Magnetic Particle
suspension at 37°C with intermittent
vortexing at setting #7 until the
particles are completely suspended.
Use PKII Buer.
Particles no longer produce
consistent results (fine brown sandy
particles and brown color are
observed in the supernatant)
20
Samples have low pH. Measure the pH of the sample and
adjust the pH to between 6 and 8.
Magnetic Particles were stored
at −20°C.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Order new materials and store them
at room temperature.
Good laboratory practices
B
Good laboratory practices for PCR and RT-PCR
•
Wear clean gloves and a clean lab coat.
–
Do not wear the same gloves and lab coat that you have previously used
when handling amplified products or preparing samples.
•
Change gloves if you suspect that they are contaminated.
•
Maintain separate areas and dedicated equipment and supplies for:
–
Sample preparation and reaction setup.
–
Amplification and analysis of products.
•
Do not bring amplified products into the reaction setup area.
•
Open and close all sample tubes carefully. Avoid splashing or spraying samples.
•
Keep reactions and components capped as much as possible.
•
Use a positive-displacement pipettor or aerosol‑resistant barrier pipette tips.
•
Clean lab benches and equipment periodically with 10% bleach solution or DNA
decontamination solution.
Avoiding false positives due to cross-contamination
To avoid false positives due to cross-contamination:
•
Prepare and close all negative control and unknown sample tubes before
pipetting the positive control.
•
Do not open tubes after amplification.
•
Use dierent sets of pipettors when pipetting negative control, unknown, and
positive control samples.
Plate layout suggestions
•
For each plate row, dispense in sequence from left to right the: negative controls,
unknown samples and ERCs, and positive controls (at the end of the row or
column).
•
Place positive controls in one of the outer columns (10–12).
•
If possible, separate all samples from each other by at least one well; if space is
limiting, place at least one well between unknown samples and controls.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
21
C
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in
the user documentation may result in personal injury or damage to the instrument
or device. Ensure that anyone using this product has received instructions in
general safety practices for laboratories and the safety information provided in this
document.
Before using an instrument or device, read and understand the safety information
·
provided in the user documentation provided by the manufacturer of the
instrument or device.
Before handling chemicals, read and understand all applicable Safety Data Sheets
·
(SDSs) and use appropriate personal protective equipment (gloves, gowns, eye
protection, and so on). To obtain SDSs, see the “Documentation and Support”
section in this document.
22
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Chemical safety
Appendix C Safety
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure
laboratory personnel read and practice the general safety guidelines for chemical
usage, storage, and waste provided below. Consult the relevant SDS for specific
precautions and instructions:
Read and understand the Safety Data Sheets (SDSs) provided by the chemical
·
manufacturer before you store, handle, or work with any chemicals or hazardous
materials. To obtain SDSs, see the "Documentation and Support" section in this
document.
Minimize contact with chemicals. Wear appropriate personal protective equipment
·
when handling chemicals (for example, safety glasses, gloves, or protective
clothing).
Minimize the inhalation of chemicals. Do not leave chemical containers open. Use
·
only with sucient ventilation (for example, fume hood).
Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
·
manufacturer cleanup procedures as recommended in the SDS.
Handle chemical wastes in a fume hood.
·
Ensure use of 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.)
After emptying a waste container, seal it with the cap provided.
·
Characterize (by analysis if needed) the waste generated by the particular
·
applications, reagents, and substrates used in your laboratory.
Ensure that the 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.
C
WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the
instrument is potentially hazardous. Follow the guidelines noted in the preceding
General Chemical Handling warning.
WARNING! 4L Reagent and Waste Bottle Safety. 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.
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
23
Appendix C Safety
C
Biological hazard safety
Biological hazard safety
WARNING! Potential Biohazard. Depending on the samples used on this
instrument, the surface may be considered a biohazard. Use appropriate
decontamination methods when working with biohazards.
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. Conduct all work in properly equipped facilities with
the appropriate safety equipment (for example, physical containment devices). Safety
equipment can also include items for personal protection, such as gloves, coats,
gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles.
Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially biohazardous materials.
Follow all applicable local, state/provincial, and/or national regulations. The following
references provide general guidelines when handling biological samples in laboratory
environment.
U.S. Department of Health and Human Services, Biosafety in Microbiological and
·
Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112,
Revised December 2009; found at:
https://www.cdc.gov/labs/pdf/CDCBiosafetymicrobiologicalBiomedicalLaboratories-2009-P.pdf
World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
·
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
24
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
Related documentation
Documentation and support
Document
PrepSEQ™ Residual DNA Sample
Preparation Kit Quick Reference
resDNASEQ™ Quantitative DNA Kits
User Guide
resDNASEQ™ Quantitative DNA Kits
Quick Reference
Thermo Scientific™ KingFisher
Flex User Manual
Applied Biosystems™ MagMAX
Express 96 User Manual
Customer and technical support
Visit thermofisher.com/support for the latest service and support information.
•
Worldwide contact telephone numbers
•
Product support information
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Product FAQs
–
Software, patches, and updates
–
Training for many applications and instruments
•
Order and web support
•
Product documentation
–
User guides, manuals, and protocols
–
Certificates of Analysis
–
Safety Data Sheets (SDSs; also known as MSDSs)
Publication
number
4469839 For brief instructions on using the
PrepSEQ™ Residual DNA Sample
Preparation Kit.
4469836 For information on performing PCR
after sample extraction.
4469837 For brief instructions on using
the resDNASEQ™ Quantitative DNA
Kits.
™
™
N07669 For information on the KingFisher
Flex instrument.
N07848 For information on the MagMAX
Express 96 DW instrument.
Description
™
™
PrepSEQ
Note: For SDSs for reagents and chemicals from other manufacturers,
contact the manufacturer.
™
Residual DNA Sample Preparation Kit User Guide
25
Documentation and support
Limited product warranty
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as
set forth in the Life Technologies' General Terms and Conditions of Sale
at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you
have any questions, please contact Life Technologies at www.thermofisher.com/
support.
26
PrepSEQ™ Residual DNA Sample Preparation Kit User Guide
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