Thermo Fisher Scientific Fast MicroSEQ User Manual
Fast MicroSEQ™ 500 16S rDNA Identification
USER GUIDE
using:
Fast MicroSEQ™ 500 16S rDNA PCR Kit and
MicroSEQ™ 500 16S rDNA Sequencing Kit
Catalog Numbers 4370489 (PCR kit) and 4346480 (Sequencing kit)
Publication Number 4393007
Revision G
For Research Use Only. Not for use in diagnostic procedures.
Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
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. 4393007
Revision Date Description
G 11 January 2021 Update for the launch of the MicroSEQ™ ID Software For SeqStudio™ Genetic Analyzer v1.0 (Cat. No.
F 31 August 2018 Update trademark, legal, and manufacturer information.
A49382).
Update the list of purification products in “Purify extension products” on page 13.
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.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
Product Information .................................................................. 5
Product information ............................................................. 5
Instrument platforms ............................................................ 5
Contents and storage ............................................................ 6
Storage guidelines .......................................................... 6
Required materials not supplied ................................................... 6
■
Methods ............................................................................... 7
Workflow ....................................................................... 7
Collect and prepare samples ..................................................... 8
Important procedural guidelines .............................................. 8
Isolate genomic DNA from samples ........................................... 8
Dilute genomic DNA for PCR ................................................. 9
Amplify the 16S rDNA region ..................................................... 9
Important procedural guidelines .............................................. 9
Prepare the PCR reactions ................................................... 9
Perform the amplification run ................................................ 10
(Optional) Analyze PCR products ............................................ 11
Purify PCR products for cycle sequencing .................................... 11
Perform cycle sequencing ....................................................... 11
Important procedural guidelines ............................................. 12
Prepare cycle sequencing reactions .......................................... 12
Perform the cycle sequencing run ............................................ 12
Purify extension products ................................................... 13
Perform electrophoresis of extension products ..................................... 14
Important procedural guidelines ............................................. 14
Configure the instrument for electrophoresis .................................. 14
Prepare samples and perform electrophoresis ................................. 15
Troubleshooting ................................................................ 16
Frequently asked questions ..................................................... 17
Sensitivity and quantitation ................................................. 17
Sample preparation and storage ............................................. 18
Contamination ............................................................ 18
Overlapping sequences .................................................... 19
PCR product size .......................................................... 19
Fast MicroSEQ
™
500 16S rDNA Identification User Guide
3
Contents
■
■
■
Species libraries ........................................................... 19
Additional documentation ................................................... 20
APPENDIX A Ordering information .............................................. 21
APPENDIX B Additional supported instruments ................................ 23
Electrophoresis settings for additional supported instruments ....................... 23
APPENDIX C Supplemental procedures and guidelines ....................... 24
Good laboratory practices for PCR and RT-PCR ................................... 24
Seal the PCR plate ............................................................. 25
Seal the plate with strip caps ................................................ 25
Seal the plate with adhesive film ............................................. 26
Prevent evaporation during electrophoresis ........................................ 26
Prepare a diluted sample ................................................... 27
Dry-down, then resuspend the sample ....................................... 27
■
APPENDIX D Supplemental product information ............................... 29
MicroSEQ™ system overview .................................................... 29
About MicroSEQ™ ID software and MicroSEQ™ ID Software For SeqStudio
Genetic Analyzer ............................................................. 29
MicroSEQ™ ID proprietary libraries ........................................... 29
Custom libraries ........................................................... 30
MicroSEQ™ ID reports ...................................................... 30
About dye-labeled terminator chemistry .......................................... 33
■
APPENDIX E Safety ............................................................... 34
Chemical safety ................................................................ 35
Biological hazard safety ......................................................... 36
■
Documentation and support ....................................................... 37
Related documentation ......................................................... 37
Customer and technical support ................................................. 37
Limited product warranty ........................................................ 38
™
4
Fast MicroSEQ™ 500 16S rDNA Identification User Guide
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product information
Product Information
The Applied Biosystems™ Fast MicroSEQ™ 500 16S rDNA PCR Kit and the Applied Biosystems
MicroSEQ™ 500 16S rDNA Sequencing Kit provide all of the reagents necessary for the amplification
and sequencing of the first 500 base pairs of the 16S ribosomal RNA gene (rDNA). The DNA sequence
of the unknown is deciphered by capillary electrophoresis on an Applied Biosystems™ Genetic Analyzer.
The FAST PCR technology used in the MicroSEQ™ PCR kits allows identification in 5 hours. MicroSEQ
ID software (3500/3500xL Genetic Analyzer) or MicroSEQ™ ID Software For SeqStudio™ Genetic
Analyzer compares the sequence to the validated MicroSEQ™ 16S rDNA 500 Library, then generates an
identification report. Variations found within the first 500 base pairs of the 16S region are sucient to
identify most bacteria to the species level.
Note: The MicroSEQ™ Full Gene 16S rDNA Identification is recommended if you need a full 16S rDNA
sequence to identify a bacterial species.
Instrument platforms
For optimum performance of the Fast MicroSEQ™ 500 16S rDNA Identification, use the:
•
Veriti™ 96‑Well Thermal Cycler
•
3500/3500xL Genetic Analyzer or SeqStudio™ Genetic Analyzer
For information on older instruments that can also be used, see Appendix B, “Additional supported
instruments”.
™
™
Fast MicroSEQ
™
500 16S rDNA Identification User Guide
5
Product Information
Contents and storage
Contents and storage
Table 1 Fast MicroSEQ™ 500 16S rDNA PCR Kit (Cat. No. 4370489)
Contents Amount Storage
FAST MicroSEQ™ 500 16S PCR Master
Mix
Positive Control, E. coli, 1 ng/µL One tube sucient for 5 positive-control assays
Negative Control, water One tube sucient for 5 negative-control assays
Table 2 MicroSEQ™ 500 16S rDNA Sequencing Kit (Cat. No. 4346480)
Contents Amount Storage
MicroSEQ™ 500 16S Forward Sequence Mix Two tubes sucient for a total of 55
MicroSEQ™ 500 16S Reverse Sequence Mix Two tubes sucient for a total of 55
One tube sucient for 50 PCR amplifications On receipt:
reactions
reactions
Storage guidelines
•
Avoid excess freeze‐thaw cycles. Aliquot reagents in smaller amounts, if necessary.
•
Before each use of the kit, allow the frozen reagents to thaw at room temperature or on ice.
IMPORTANT! Do not heat the reagents.
•
Whenever possible, keep thawed reagents on ice during use.
•
Mix the reagents by gently vortexing the tubes. Centrifuge the tubes briefly to collect all liquid at
the bottom of the tube.
–25°C to –15°C
After first use:
2–8°C in a PCR clean
room
–25°C to –15°C
Required materials not supplied
Contact your local MicroSEQ™ ID representative for a list of additional materials and equipment
required.
6
Fast MicroSEQ™ 500 16S rDNA Identification User Guide
Workflow
Harvest bacterial colony, isolate DNA, then
Methods
Collect and prepare samples
dilute DNA for PCR
q
Amplify DNA
Prepare reactions, perform amplification, analyze PCR
products (optional), then purify PCR products
q
Perform cycle sequencing
Prepare reactions, perform cycle sequencing, then purify
extension products
q
Perform electrophoresis
Configure instrument, then prepare and run samples
q
Analyze data
Veriti™ 96‑Well
Thermal Cycler
Veriti™ 96‑Well
Thermal Cycler
SeqStudio™ Genetic
Analyzer or
3500/3500xL Genetic
Analyzer
Fast MicroSEQ™ 500 16S rDNA Identification User Guide
7
Methods
Collect and prepare samples
Collect and prepare samples
Important procedural guidelines
•
Review “Good laboratory practices for PCR and RT-PCR” on page 24.
•
When the isolated DNA (in PrepMan™ Ultra supernatant) is not in use, store it at −15 to −25°C .
Before use, thaw, then vortex and centrifuge the stored supernatant. Alternatively, cover and store
the supernatant at 4°C for up to 1 month.
Isolate genomic DNA from samples
Isolate bacterial genomic DNA from bacterial colonies using PrepMan™ Ultra Sample Preparation
Reagent. See the PrepMan™ Ultra Sample Preparation Reagent Protocol for additional information.
1.
Obtain the sample, then add PrepMan™ Ultra Sample Preparation Reagent:
If starting from a ...
Culture broth
Culture plate
IMPORTANT! The ideal colony size is 2–3 mm. For smaller colonies, decrease the amount of
PrepMan™ Ultra Sample Preparation Reagent to 50 μL from the 100 μL suggested in the protocol.
2.
Vortex the sample for 10 to 30 seconds.
3.
Heat the sample for 10 minutes at 100°C in a heat block, then cool the sample to room
temperature for 2 minutes.
Follow this procedure ...
1.
Pipet 1 mL of culture broth (containing less than 107 cfu/mL of bacteria) into a new
2-mL screw-cap microcentrifuge tube or any other microcentrifuge tube that can be
tightly closed.
2.
Centrifuge the sample for 2 minutes in a microcentrifuge at maximum speed. Aspirate
and discard the supernatant.
3.
Add 100 µL of PrepMan™ Ultra Sample Preparation Reagent, then close the cap tightly.
1.
Select a small sample amount (2–3 mm) from an isolated colony by using a 1 µL loop
or the straight end of a 1 µL loop.
2.
Suspend the cells in 100 µL of PrepMan™ Ultra Sample Preparation Reagent in a 2-mL
screw-cap microcentrifuge tube or any other microcentrifuge tube that can be tightly
closed.
4.
Centrifuge the sample for 2 minutes in a microcentrifuge at maximum speed.
5.
Transfer 50 µL of the supernatant into a new microcentrifuge tube.
8
Fast MicroSEQ™ 500 16S rDNA Identification User Guide
Dilute genomic DNA for PCR
1.
Pipet 495 μL of nuclease‐free water into a 1.5‐mL microcentrifuge tube.
2.
Add 5 μL of the PrepMan™ Ultra supernatant to obtain a 1:100 dilution.
Note: For samples with low biomass, make a smaller dilution (for example, use 195 μL of
nuclease-free water to make a 1:40 dilution). The minimum recommended dilution for the
PrepMan™ Ultra supernatant is 1:10.
Note: If the PrepMan™ Ultra supernatant is colored (typically a shade of black or green), PCR
inhibition may occur. See “Troubleshooting” on page 16.
Amplify the 16S rDNA region
Important procedural guidelines
•
Select the appropriate tubes or 96‐well plates for your thermal cycler. See your instrument user
guide (available at thermofisher.com).
•
Using strip caps instead of 96-well adhesive plate covers may help reduce cross-contamination.
•
Before preparing the PCR reactions, review “Good laboratory practices for PCR and RT-PCR” on
page 24 and “Storage guidelines” on page 6 for sample and reagent handling instructions.
•
If necessary after performing PCR or purifying PCR products, cover and store the PCR products at
–15°C to –25°C until you are ready to use them.
Methods
Amplify the 16S rDNA region
Note: PCR products are stable for 6 months or longer at –15°C to –25°C.
Prepare the PCR reactions
1.
Vortex the diluted supernatant to mix the tube contents.
2.
Using the volumes that are shown in the table, prepare samples and controls in MicroAmp
reaction tubes or 96‐well plates.
Reaction type
Negative controls
Samples
Positive controls
Volume for one reaction
•
15 μL FAST PCR Master Mix
•
15 μL negative control (provided with kit)
•
15 μL FAST PCR Master Mix
•
15 μL of 1:100 dilution of PrepMan® Ultra supernatant
•
15 μL FAST PCR Master Mix
•
15 μL positive-control DNA (provided with kit)
™
Fast MicroSEQ™ 500 16S rDNA Identification User Guide
9
Methods
Amplify the 16S rDNA region
Note: To help avoid cross‐contamination, we recommend that you pipet components in the
following order: negative controls, samples, positive controls. If possible, leave empty cells
between dierent reaction types.
3.
Use strip caps and the capping tool, or adhesive film and the sealing tool, to cap the tubes or plate
(see “Seal the PCR plate” on page 25). Vortex, centrifuge briefly, then place the tubes or the plate
in the thermal cycler.
IMPORTANT! Apply significant downward pressure on the sealing tool in all steps to form a
complete seal.
Perform the amplification run
1.
Set the appropriate ramp mode for your thermal cycler:
•
Veriti™ 96‑Well Thermal Cycler—Default
Note: To use the default mode, select Browse/New Methods4New, then edit the thermal
cycling conditions. See the Veriti™ 96‑Well Thermal Cycler User Guide for details.
•
(3500/3500xL only) 9800 Fast Thermal Cycler—Fast (F96)
•
(3500/3500xL only) GeneAmp™ PCR System 9700—Maximum (Max)
2.
Set the thermal cycling conditions:
Initial step
HOLD CYCLE HOLD HOLD
95°C
10 seconds
3.
Set the reaction volume to 30 μL, then start the run.
4.
Before removing the caps or adhesive film, briefly centrifuge the tubes or plate.
Each of 30 cycles
Melt Anneal
95°C
0 seconds
15 seconds
64°C
Final extension Final step
72°C
1 minute
Note: Centrifuging helps avoid cross‐contamination from liquid remaining on the caps or plate
covers.
4°C
∞
10
Fast MicroSEQ™ 500 16S rDNA Identification User Guide
(Optional) Analyze PCR products
Analyze PCR products to confirm the presence of amplified DNA, or to estimate the PCR product yield.
The cycle-sequencing protocol works best with 5 to 20 ng of amplicon input.
1.
Load 5 μL of PCR product per lane on a 2% agarose gel separation (such as E‑Gel™ available from
thermofisher.com), or prepare your own gel.
2.
Use the Mass Standard Ladder to estimate the PCR product yield. In a positive control or sample,
a single fragment ranging from 460 to 560 bp in size should be detected on a gel. Actual fragment
size depends on the bacterial species. No product should be visible in a negative control reaction.
Methods
Perform cycle sequencing
IMPORTANT! If your samples show no PCR product, PCR inhibition is the most likely cause. See
“Troubleshooting” on page 16.
Purify PCR products for cycle sequencing
Remove unused dNTPs and primers from each PCR product with ExoSAP-IT™ Express PCR Product
Cleanup Reagent (Cat. No. 75001).
IMPORTANT!
product literature.
Follow the guidelines for the starting sample volume for cleanup as directed in the
Perform cycle sequencing
Cycle sequencing occurs when successive rounds of denaturation, primer annealing, and primer
extension in a thermal cycler result in the incorporation of dye terminators into extension products.
The products are then loaded into a genetic analyzer to determine the 16S rDNA sequence. For
additional information about cycle sequencing chemistries, refer to the DNA Sequencing by Capillary
Electrophoresis Chemistry Guide.
Fast MicroSEQ™ 500 16S rDNA Identification User Guide
11
Methods
Perform cycle sequencing
Important procedural guidelines
•
Select the appropriate tubes or 96‐well plates for your thermal cycler. See your instrument user
guide (available at thermofisher.com).
•
Using strip caps instead of 96‐well adhesive plate covers may help reduce cross-contamination.
•
If you are using a CentriSep™ Spin Column to purify extension products (see “Purifying Extension
Products” on page 15), hydrate the column with highly purified (nuclease free) water during the
cycle sequencing run.
•
If necessary, cover and store the unused portions of the purified PCR products at –15°C to –25°C
until you are ready to use them.
Note: PCR products are stable for 6 months or longer at –15°C to –25°C.
•
If necessary, cover and store the extension products at 4°C overnight or at –15°C to –25°C for up
to 1 week before purifying them.
Prepare cycle sequencing reactions
1.
Before you remove the tube or plate caps, briefly centrifuge the purified PCR products.
Note: Centrifuging helps avoid cross‐contamination from liquid remaining on the caps or plate
covers.
2.
In reaction tubes or a 96‐well plate, prepare separate forward‐ and reverse-sequencing reactions
for each PCR product and control:
•
Forward‐sequencing reaction—Combine 7 μL of purified PCR product or control with 13 μL
forward sequence mix.
•
Reverse‐sequencing reaction—Combine 7 μL of purified PCR product or control with 13 μL
reverse sequence mix.
Note: To help avoid cross‐contamination, pipet components in the following order: negative
controls, samples, positive controls.
Perform the cycle sequencing run
1.
Cap the tubes or the plate, then place the tubes or the plate in the thermal cycler.
2.
Set the appropriate ramp mode for your thermal cycler:
•
Veriti™ 96‑Well Thermal Cycler—Default
•
(3500/3500xL only) 9800 Fast Thermal Cycler—Fast (F96)
•
(3500/3500xL only) GeneAmp™ PCR System 9700—Maximum (Max)
12
Fast MicroSEQ™ 500 16S rDNA Identification User Guide
Loading...