Bioline ISOLATE II Plant DNA Kit, Product Manual

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Plant DNA Kit
13
8.2 SUPPORT PROTOCOL FOR PURIFYING FUNGAL GENOMIC DNA
Additional reagents/components to be supplied by the user:
•
Ethanol (96–100%)
• Chloroform
• Micropestle
•
Siliconized glass beads or sea sand
1 Homogenize sample
Wash 50–200 mg mycelium (fresh weight) or 50‑200 mg material from a fruiting body
of macro fungi in ethanol. Mycelium can be obtained from a liquid culture or scraped
off (with or without agar) from the surface of a solid medium.
Submerge sample completely in ethanol and mix carefully. In most cases, short
washes in ethanol are sufficient; however, overnight incubation can sometimes
increase DNA yield (long‑term storage in ethanol is also possible). Remove ethanol by
pipetting and squeezing the sample.
2
Cell lysis
Transfer sample into a 1.5 mL microcentrifuge tube (not supplied). Add 150 mg
siliconized glass beads or sea sand and 200 μL Lysis Buffer PA1. Homogenize sample
using a micropestle and vortex regularly. Add additional 100 μL Lysis Buffer PA1 and
continue to homogenize the sample.
Note: If the sample cannot be easily handled because e.g. the sample material soaks up too
much buffer, additional Lysis Buffer PA1 can be added. The volume of Binding Buffer PB (step 4)
however, has to be increased proportionally.
Optional: If the sample is rich in RNA or protein, we recommend adding 10 μL RNase A and/or
Proteinase K (5–10 mg/mL stock solution, see ordering information), respectively, to the PA1 lysis
solution in order to minimize contaminants.
Incubate for 10 min at 65°C.
Note: For some fungi it might be advantageous to increase the incubation time to 30–60 min.
Add 100 μL chloroform. Vortex for 10s and separate phases by centrifugation for 15
min at 20,000 x g. Pipette the top aqueous layer into a new 1.5 mL microcentrifuge
tube (not supplied).
3 Filter lysate
Place ISOLATE II Filter in Collection Tube (2 mL), apply lysate and centrifuge for 1 min
at 11,000 x g. This step reduces solution turbidity and viscosity.
If a visible pellet forms, transfer supernatant whilst avoiding the pellet to a new 1.5 mL
microcentrifuge tube (not supplied).
Alternatively, pass lysate ≥5 times through a 20 gauge (0.9 mm) needle and syringe.
Proceed with step 3 from section 8.1.