Bead Beating: A Primer (continued)
PLANTS
Pollen Homogenization
Pollen has a rigid cell wall that is most
efficiently disrupted with zirconium beads. As the size range of pollen is
very broad, bead size must be matched to the type of pollen. Undersized
beads will have insufficient mass to crack open pollen, while using large
beads will result in too few collisions. Pollen less than 10
µm in size should be disrupted with 200 µm zirconium beads, while pollen
10-50 µm in size should be processed with 400 µm zirconium beads. Large
pollen (=50 µm) is most effectively homogenized with 800 µm zirconium beads.
Depending upon the bead beater, samples can be processed in disruption
tubes, deep well plates, 4 ml polycarbonate or polyethylene vials, and 15 ml
polycarbonate vials.
Procedures
| 1. |
Pollen
can be effectively disrupted by bead beating, though the choice of beads is
dependent on the size of the pollen granules. Pollen can
be collected as a dry powder and weighed for processing.
The beads used to bead beat pollen are 100 µm zirconium (for pollen <10 µm),
400 µm zirconium (for pollen 10-50 µm), and 800 µm zirconium (for pollen
larger than 50 µm). |
| 2. |
Prepare samples according to the following table: |
|
Disruption Tubes (2 ml) |
Deep Well Plates (square wells) |
4 ml Vials |
15 ml Vials |
| Sample Mass |
10 mg |
10 mg |
Up to 25 mg |
Up to 100 mg |
| Buffer Volume |
500 µl |
500 µl |
1 ml |
5 ml |
| Bead Volume |
400 µl |
300 µl |
1 ml |
3 ml |
| Processing Time |
2-5 minutes |
2-5 minutes |
5 minutes |
5-10 minutes |
| Bead Beater Speed |
High |
High |
High |
High |
| 3. |
Process the samples as stated in the table. For heat sensitive samples, the processing can be divided into short
bursts followed by a cooling period. |
| 4. |
Ruptured pollen grains appear with a crack or split.
Visual inspection can be used to estimate homogenization efficiency
using a microscope (number of cracked grains/total grains in the field of
view). If the efficiency is less than 60%, continue to
homogenize the sample. |
| 5. |
Lysate can then be processed as needed. |
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