Thermo Fisher Scientific MicroSeq ™ D2 LSU rDNA Fungal Sequencing Kit Quick Start

Brand: Thermo Fisher Scientific

Model: MicroSeq ™ D2 LSU rDNA Fungal Sequencing Kit

Type: Quick Start

MicroSeq™ D2 LSU rDNA Fungal Sequencing Kit Quick Start Card

Negative

Controls • 25 µL PCR Master Mix

• 25 µL Negative Control water

• 25 µL PCR Master Mix

• 25 µL Diluted Positive Control DNA

(1 µL DNA + 24 µL H2O)

Positive

Controls

• 25 µL PCR Master Mix

• 25 µL diluted genomic DNA

preparation (see reverse side)

Samples

1. Preparing PCR Samples (10 min.) 2. PCR (2.25 hrs.)

3. Purifying PCR Products (45 min.)

4. Preparing Sequencing Reactions (10 min.)

{

Forward • 3 µL purified PCR product

• 4 µL deionized water

• 13 µL Forward Sequencing Mix

• 3 µL purified PCR product

• 4 µL deionized water

• 13 µL Reverse Sequencing Mix

ABI PRISM® 310 Genetic Analyzer: See page 25 of the protocol.

ABI PRISM® 377 DNA Sequencer: See page 26 of the protocol.

Entire sequencing reaction

(add to center of gel)

Reverse

{

6. Purifying Extension Products (30 min. not including hydration time)

7. Electrophoresis

Fungal Sample Preparation (PrepMan™ and FastPrep™ Method)

FastPrep™ is a trademark of BIO101

P/N 4312443 Rev. A

Purified

PCR

product

(use 3 µL in

sequencing

rxns)

20 µL deionized water

wait 1 min.

Spin 3 min. @

1000 x

Keep tube and

discard column

New labeled

collection tube

Invert column

Spin 6 min. @

500 x

Discard

filtrate

Spin 15 min. @

500 x

Discard collection

tube and contents

400 µL deionized water

+ entire PCR product

500 µL deionized

water

Microcon-100

column

Collection

tube

10 min.

95°C 30 sec.

95°C 35 cycles

30 sec.

53°C

1 min.

72°C 10 min.

72°C

forever

4°C

Rapid thermal ramp

(1°C/sec.) between steps

10 sec.

96°C 25 cycles

5 sec.

50°C

4 min.

60°C

forever

4°C

Rapid thermal ramp

(1°C/sec.) between steps

Dry sample in vacuum

centrifuge 15 min. or

until dry

Spin

column

Add 0.8 mL

deionized water and mix

Hydrate 30 min.

Remove caps and drain

Insert into wash tube

Spin 2 min. @ 750 x

Insert column into

sample collection tube

Spin 2 min. @ 750 x

Keep tube and discard

column

Possible Stopping Points = (keep at 4°C)

5. Cycle Sequencing (2.5 hrs.)

Small loopful of yeast cells

or edge of filamentous colony

Small loopful of yeast cells

or edge of filamentous colony

(Many yeasts and some filamentous

fungi are easily prepared for PCR –

use this method initially.)

Standard FastPrep™

tube + cap

Suspend in 200 µL of

PrepMan solution.

• Incubate tube at 100˚C for 10 min.

• Vortex briefly.

Centrifuge briefly to settle cell

debris (2 – 5 sec.).

Process 20 sec. @ setting 4.5 in

FastPrep instrument.

Suspend in 1mL CLS-Y buffer +

garnet powder + 1/4" sphere

according to manufacturer's

instructions.

Follow standard SPIN FILTER protocol

in the FastPrep Manual for DNA

isolation.

Elute DNA with 100µL DES.

For PCRs, combine:

Example:

• 1 µL of supernatant from either extraction method

• 24 µL deionized water

To minimize potential pipetting error you can prepare a larger volume using any

multiple of the above (water for larger dilution is not included in the kit). Example:

• 10 µL DNA sample

• 240 µL deionized H2O

For limited label licence information, see this product's

package insert or protocol book.

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

PrepMan™

Yeasts and Filamentous Fungi FastPrep™

Resistant Yeasts and Filamentous Fungi

(Those which fail PrepMan™ – Use this method)

After extraction either:

1) Transfer supernatant to new

microcentrifuge tube and store frozen.

2) Store tube and contents frozen.

Thermo Fisher Scientific MicroSeq ™ D2 LSU rDNA Fungal Sequencing Kit Quick Start

Thermo Fisher Scientific MicroSeq ™ D2 LSU rDNA Fungal Sequencing Kit Quick Start

Thermo Fisher Scientific MicroSeq ™ D2 LSU rDNA Fungal Sequencing Kit Quick Start

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