Bioline ISOLATE II Plant DNA Kit, Product Manual

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Product Manual www.bioline.com/isolate
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8.3 SUPPORT PROTOCOL FOR DUNG, ANIMAL FECAL, SOIL AND COMPOST SAMPLES
Additional reagents/components to be supplied by user:
•
Mortar and pestle or bead mill homogenizer
• Sea sand (siliconized)
• Extraction buffer: 2 M NaCl, 20 mM EDTA, 100 mM Tris/HCl, 2% (w/v) CTAB, 2% (w/v)
polyvinylpyrrolidone (MW 40,000), pH 8.0
1
Homogenize sample
In a petri dish, weigh 5 g soil or 2 g fecal sample and add extraction buffer until
sample is completely soaked. Heat in a microwave oven (400W) for several seconds
until extraction buffer foams.
Extraction buffer may be added to maintain the sample in a slushy state.
2 Cell lysis
Transfer sample into a bead mill or mortar. Add 0.5 mL sand and disrupt the sample
(see step 2 from section 8.2 for sample lysis/homogenization details).
3 Filtration / Clarification of lysate
Transfer the homogenized sample into a centrifuge tube and centrifuge for 10 min at
5,000 x g. Pipette 300 μL of the clear supernatant into a new 1.5 mL microcentrifuge
tube (not supplied).
Proceed with step 3 (filter crude lysate) from section 8.1.
9. TROUBLESHOOTING GUIDE
CLOGGED COLUMNS
POSSIBLE CAUSE RECOMMENDED SOLUTION
Too much sample material Do not use more sample material than recommended in the
protocol. Centrifuge large amounts of sample material before
loading it onto the ISOLATE II Filter column.
Make sure the cleared lysate is absolutely free of suspended matter
before loading it onto the ISOLATE II Plant DNA Spin Column.
Increase centrifugation speed and time.
Increase volume of Lysis Buffer PA1 or PA2.
Incomplete lysis Increase volume of Lysis Buffer PA1 or PA2.
Increase incubation time to overnight.
DEGRADED DNA
POSSIBLE CAUSE RECOMMENDED SOLUTION
Sample contaminated with DNase If another elution buffer than Elution Buffer PG is used, ensure it
is free of DNase activity, for example addition of 1 mM EDTA or
heating the buffer to 70°C for 10 min.