MicroSeq Full Gene 16S rDNA Bacterial Identification Kit

MicroSeq Full Gene

16S rDNA Bacterial Identification Kit
For safety guidelines, refer to the Safety section in the Preface of the MicroSeq Full Gene 16S rDNA Bacterial
Identification Kits Protocol (PN 4346292). For all chemicals in bold type, read the MSDS and follow the handling
instructions. Wear appropriate eyewear, clothing, and gloves.

Summary
The MicroSeq Full Gene 16S rDNA Bacterial Identification PCR Kit (PN 4349161) and the MicroSeq Full Gene
16S rDNA Bacterial Identification Sequencing Kit (PN 4347483) provide all the reagents necessary to sequence
the 16S ribosomal RNA bacterial gene (16S rDNA). The resulting DNA sequence is analyzed and compared to a
library of 16S rDNA bacterial gene sequences using MicroSeqID Analysis Software.
This quick reference card provides simplified procedures for using the MicroSeq Full Gene bacterial identification
kits. The MicroSeq Full Gene 16S rDNA Bacterial Identification Kits Protocol provides detailed procedural
information.

Kit Contents
The MicroSeq Full Gene 16S rDNA Bacterial Identification system has two kits: PCR and Sequencing. The
following table describes the components of each kit.
Kit
MicroSeq Full Gene 16S
rDNA Bacterial
Identification PCR Kit
(PN 4349161)

MicroSeq Full Gene 16S

rDNA Bacterial
Identification Sequencing
Kit (PN 4347483)

Component
PCR Master Mixes

Description
Three different tubes containing 2 PCR Master Mix
Each tube is designed to amplify a specific portion of the
16S rRNA gene sequence from bacterial genomic DNA.
Each tube contains enough PCR Master Mix to perform
10 PCR amplifications, 5 negative control assays, and 5
positive control assays.

Positive Control DNA

One tube of Positive Control DNA at 1 ng/L

Negative Control (Water)

One tube of Negative Control

Forward Sequencing Master

Mixes

Three different tubes containing enough mix to perform a

total of 15 reactions (10 sequencing and 5 control
reactions)
Each tube corresponds to one of the PCR Master Mix
tubes from the PCR kit.

Reverse Sequencing Master

Mixes

Three different tubes containing enough mix to perform a

total of 15 reactions (10 sequencing and 5 control
reactions)
Each tube corresponds to one of the PCR Master Mix
tubes from the PCR kit.

QUICK REFERENCE CARD

Process
Indicates possible stopping points.

Isolate bacterial genomic

DNA.

Isolate bacterial genomic DNA using the PrepMan Ultra Sample

Preparation Reagent (PN 4322547).
Store the supernatant at 20 C.
Refer to the PrepMan Ultra Sample Preparation Reagent Protocol
(PN 4318925) or the MicroSeq Full Gene 16S rDNA Bacterial Identification
Kits Protocol (PN 4346292) for more information.

Make the working stock

of DNA.

a. Pipette 495 L of nuclease-free water (Ambion PN 9937) into a 1.5-mL

microcentrifuge tube (Ambion PN 12450).
b. Add 5 L of the PrepMan Ultra supernatant to get a 1:100 dilution.
c. Vortex the tube to mix the solution.
d. Store the remaining bacterial supernatant at 20 C.

Prepare samples for PCR.

The PCR kit is optimized for use with 25 ng of bacterial genomic DNA.
Note: If necessary, make additional dilutions of the working stock before
proceeding to PCR to minimize the effects of PCR inhibition.
If preparing...

Then combine...

Negative Controls

15 L 2 PCR Master Mix

15 L nuclease-free water

Positive Controls

15 L 2 PCR Master Mix

15 L of the E. coli positive-control DNA

Samples

15 L 2 PCR Master Mix

15 L of the working stock (1:100 dilution)

Note: You should have three tubes for each sample,

one for each of the three PCR Master Mixes in the
MicroSeq Full Gene PCR kit.
Cap the tubes or seal the 96-well tray, then place them in the thermal cycler.

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Perform PCR.

a. Program the 9700 thermal cycler in 9600 emulation mode, as follows:

Initial
Step

Each of 30 Cycles
Melt

HOLD
95 C
10 min

GeneAmp

Anneal

Final
Step

HOLD

HOLD

72 C
10 min

4 C

Extend

CYCLE
95 C
30 sec

Final
Extension

60 C
30 sec

72 C
45 sec

. The initial 10-min, 95 C heating step is required to activate the AmpliTaq Gold
DNA Polymerase.
. You can increase the number of cycles to increase the PCR yield, but doing so can
cause additional background signal from the negative control.

b. Set the reaction volume for thermal cycling to 30 L, then start the run.
Sample

Fragment 1

Fragment 2

Fragment 3

PCR
Master Mix
1

PCR
Master MIx
2

PCR
Master MIx
3

PCR
Product
1

PCR
Product
2

PCR
Product
3

c. Store the PCR products at 15 to 25 C until you are ready to use them.

Analyze PCR products.

DNA Mass
Ladder

Positive
Control 1

Positive
Control 2

Positive
Control 3

2.0kb

Alternatively, run a 2% E-Gel (Invitrogen G5000-02). Read and understand the

MSDS provided by the chemical manufacturer.

1.2kb

The positive control and samples should display three PCR products: 1 band
460 to 560 bp and 2 bands 700800 bp. The product sizes can vary,
depending on the bacterial species. No product should be visible for the
negative control.

0.8kb

0.4kb

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GR2291

0.2kb

0.1kb

Determine if a PCR product is present in your samples by running 10 L of

your sample on a 2% agarose gel. Loading 10 L of the PCR product per lane
is sufficient to detect amplified DNA with ethidium bromide staining.

Note: If your samples show no PCR product, PCR inhibition is the most likely
cause. For information, see the Troubleshooting section in the MicroSeq Full
Gene 16S rDNA Bacterial Identification Kits Protocol.

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Purify PCR products.

After gel analysis, you have 20 L of PCR product mixture. Remove unused
dNTPs and primers from the PCR product mixture using one of the following
products recommended by Applied Biosystems:

ExoSAP-IT (USB PN 78200; http://www.usbweb.com). Read and

understand the MSDS provided by the chemical manufacturer.
Montage PCR Filter Unit (Millipore PN UFC7 PCR50)

Be sure you follow guidelines for the starting sample volume for purification as
directed in the product literature.

Prepare cycle sequencing

reactions.

PCR
Product
1

PCR
Product
2

PCR
Product
3

Purified
PCR
1

Purified
PCR
2

Purified
PCR
3

a. In 0.2-mL microcentrifuge tubes or a 96-well tray, combine in parallel for

each PCR product:
7 L of the purified PCR product and 13 L of the forward sequencing
master mix
7 L of the purified PCR product and 13 L of the reverse sequencing
master mix
b. Cap the tubes or seal the 96-well tray, then place them in the thermal
cycler.
IMPORTANT! Make sure that you use the Forward and Reverse Sequencing
Master Mixes for the corresponding PCR Master Mix that was used in the
reaction.
Note: After the amplification step, you had three tubes per sample (one for
each of three PCR Master Mixes). In this step, preparing a forward and reverse
sequencing reaction for each of the three tubes, brings the total number of
tubes per sample to six.

Purified
PCR
1

Reverse 1

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Forward 1

Purified
PCR
2

Reverse 2

Forward 2

Purified
PCR
3

Reverse 3

Forward 3

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Perform cycle
sequencing.

a. Program the 9700 thermal cycler in 9600 emulation mode using the
following thermal cycling conditions:
Each of 25 Cycles
Melt

Anneal

Final
Step

Extend

CYCLE
96 C
10 sec

GeneAmp

50 C
5 sec

HOLD
60 C
4 min

4 C

b. Set the reaction volume for thermal cycling to 20 L, then start the run.
c. Store the extension products at 4 C overnight or 20 C for up to 1 week.

Purify extension products.

After cycle sequencing, remove excess dye terminators and primers from the
cycle sequencing reaction using one of the following products:
Applied Biosystems
recommends using...

If you performed PCR in...

Microcentrifuge tubes

DyeEx 2.0 Spin Kit (Qiagen PN 63204)

96-well plates

DyeEx 96 Kit (Qiagen PN 63181)

Follow the guidelines and procedures that come with the kits.

10

Perform electrophoresis
using one of the following
instruments:

3730/3730xl Instrument

3100/3100-Avant Instrument

a. Make sure that the instrument is configured using the parameters in the
following table:

Instrument

Filter
Set

310

DyeSet/Primer
(Mobility File)

Run Module

Basecaller

Seq POP6
(1 mL)E

KB.bcp

KB_310_POP6_
BDTv1_
50Std.mob

3100
3100-Avant

StdSeq50_
POP6_1

KB.bcp

KB_3100_POP6_
BDTv1.mob

3730
3730xl

LongSeq50_
POP7

KB.bcp

KB_3730_POP7_
BDTv1.mob

. Applied Biosystems recommends that you use the basecaller and mobility file names
listed in this table with the Data Collection 2.0 (DC 2.0) Software. Certain basecaller and
mobility files issued for previous releases of the software are compatible with the DC
2.0 software. Refer to the MicroSeqID Analysis Software Online Help for more
information about naming conventions for basecaller and dyeset/primer files.
ABI PRISM

310 Instrument

IMPORTANT! You must use the 50-cm capillary array length regardless of the
instrument you use.
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10

(continued)

b. Spin down the microcentrifuge tubes containing the purified extension

products in a speed vac.
c. Resuspend the DNA in 15 L Hi-Di Formamide (PN 4311320).
d. Perform the sequencing run.
e. Export the sequencing files to the MicroSeqID Analysis Software.
IMPORTANT! Resuspending in formamide is the recommended loading
procedure. Alternatively, you can directly load 10 to 20 L of the extension
products onto the instruments. This procedure, however, might require
optimization. For example, you might have to adjust injection times if the signal
strength is too high.

Copyright 2004, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. For
limited license information about this kit, please see the MicroSeq Full Gene 16S rDNA Bacterial Identification Kit Protocol
(PN 4346292).
Applied Biosystems and MicroSeq are registered trademarks and AB (Design), Applera, Hi-Di, and PrepMan are trademarks of
Applera Corporation or its subsidiaries in the US and/or certain other countries.
AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems, Inc.
All other trademarks are the sole property of their respective owners.
Printed 03/2004

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Part Number 4346297 Rev. A