Thermo Fisher Scientific Fast MicroSEQ Quick Reference

QUICK REFERENCE

Fast MicroSEQ™ 500 16S rDNA Identification

Catalog Numbers 4370489 (PCR kit) and 4346480 (Sequencing kit)

Pub. No. 4393012 Rev. D

Note: For safety and biohazard guidelines, see the “Safety” appendix in the Fast MicroSEQ™ 500 16S rDNA Identification User Guide
(Pub. No. 4393007). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves.

Procedures

Isolate genomic DNA from

1

samples

a. Obtain the sample, then add PrepMan™ Ultra Sample Preparation Reagent:

If starting from a ... Follow this procedure ...

Culture broth

Culture plate

b. Vortex the sample for 10 to 30 seconds.

c. Heat the sample for 10 minutes at 100°C in a heat block, then cool the sample to room

temperature for 2 minutes.

d. Centrifuge the sample for 2 minutes in a microcentrifuge at maximum speed.

e. Transfer 50 µL of the supernatant into a new microcentrifuge tube.

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.

Dilute genomic DNA for

2

PCR

a. Pipet 495 μL of nuclease‐free water into a 1.5‐mL microcentrifuge tube.

b. Add 5 μL of the PrepMan™ Ultra supernatant to obtain a 1:100 dilution.

Note: If the PrepMan™ Ultra supernatant is colored (typically a shade of black or green), see

“Troubleshooting” in the Fast MicroSEQ™ 500 16S rDNA Identification User Guide.

For Research Use Only. Not for use in diagnostic procedures.

Prepare the PCR reactions

3

a. Vortex the diluted supernatant to mix the tube contents.

b. Using the volumes that are shown in the table, prepare samples and controls in MicroAmp

reaction tubes or 96‐well plates.

Reaction type Volume for one reaction

Negative controls

• 15 μL FAST PCR Master Mix

• 15 μL negative control (provided with kit)

™

Perform the amplification

4

run

Samples

Positive controls

c. Use strip caps and the capping tool, or adhesive film and the sealing tool, to cap the tubes or

plate. Vortex, centrifuge briefly, then place the tubes or the plate in the thermal cycler.

a. 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)

b. Set the thermal cycling conditions:

Initial step

HOLD CYCLE HOLD HOLD

95°C

10 seconds

c. Set the reaction volume to 30 μL, then start the run.

Melt Anneal

95°C

0 seconds

• 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)

Each of 30 cycles

64°C

15 seconds

Final extension Final step

72°C

1 minute

4°C

∞

(Optional) Analyze PCR

5

products

Purify PCR products for

6

cycle sequencing

Prepare cycle sequencing

7

reactions

d. Before removing the caps or adhesive film, briefly centrifuge the tubes or plate.

a. 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.

b. 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.

Remove unused dNTPs and primers from each PCR product with ExoSAP-IT™ Express PCR Product
Cleanup Reagent (Cat. No. 75001).

a. Before you remove the tube or plate caps, briefly centrifuge the purified PCR products.

b. 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.

2 Fast MicroSEQ

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500 16S rDNA Identification Quick Reference