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.)
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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
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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
g
Keep tube and
discard column
New labeled
collection tube
Invert column
Spin 6 min. @
500 x
g
Discard
filtrate
Spin 15 min. @
500 x
g
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
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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
g
Insert column into
sample collection tube
Spin 2 min. @ 750 x
g
Keep tube and discard
column
Possible Stopping Points = (keep at 4°C)
5. Cycle Sequencing (2.5 hrs.)
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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)
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After extraction either:
1) Transfer supernatant to new
microcentrifuge tube and store frozen.
2) Store tube and contents frozen.
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