Bioline ISOLATE II Plant DNA Kit, Product Manual

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Plant DNA Kit
7
6. EQUIPMENT AND REAGENTS TO BE SUPPLIED BY USER
When working with chemicals, always wear a suitable lab coat, protective goggles and
disposable gloves. Please consult the relevant MSDS from the product supplier for further
information.
•
96–100% ethanol
†
(for Wash Buffer PAW2)
• Equipment for sample disruption and homogenization (section 7.2). One or more of
the following are required depending on chosen method.
o Mortar and pestle
o Rotor‑stator or bead‑mill homogenizer
o Vortex mixer
o Liquid nitrogen
• Microcentrifuge tubes (1.5 mL)
• Disposable tips
• Pipettes
•
Microcentrifuge (capable of 11,000 x g)
• Thermal heating block or water bath
†
Molecular biology grade ethanol is recommended. Do not use denatured alcohol which contains
unwanted additives such as methanol and acetone.
7. IMPORTANT NOTES
7.1 HANDLING AND STORING STARTING MATERIALS
Plant samples can be stored in ethanol, lyophilized/dried or frozen. Fresh material can be
kept at 4°C for 24 hours but should be frozen at ‑20°C for longer storage.
7.2 DISRUPTING AND HOMOGENIZING STARTING MATERIALS
Due to the tough nature of plant tissue, the lysis procedure is most effective with
well‑homogenized, powdered samples. Suitable methods include commercial rotor‑stator
homogenizers or bead mills using steel or glass beads. However, we recommend grinding
with a mortar and pestle in the presence of liquid nitrogen to obtain optimal yields. After
homogenization and treatment of the sample with lysis buffer, the crude lysate can be cleared
easily either with an ISOLATE II Filter or by centrifugation.
Disruption and homogenization using a mortar and pestle
A mortar and pestle can be used in combination with liquid nitrogen to disrupt and lyse frozen
plant tissue samples. Grind the frozen tissue into a fine powder and add liquid nitrogen as
necessary. It is important to ensure the sample does not thaw during or after grinding. Then
transfer tissue powder with a precooled spatula into a liquid nitrogen cooled tube and allow
liquid nitrogen to evaporate before closing the tube.