Bioline ISOLATE II Plant DNA Kit, Product Manual

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Product Manual www.bioline.com/isolate
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Disruption and homogenization using a bead mill homogenizer
Put 4‑5 beads (e.g. 7 mm diameter steel beads) and plant material into a 15 mL Falcon tube.
Chill the tube in liquid nitrogen and vortex for approx. 30 seconds. Repeat the cooling and
vortexing procedure until all plant material is ground to a fine powder. Chill the tube once
more and remove the beads by gently rolling them out or remove with a magnet. Keep the
material frozen throughout the whole homogenization procedure. Nitrogen should not be
added to the tube as this leads to sticking and loss of plant material attached to the beads.
Disruption and homogenization using a rotor‑stator homogenizer
Rotor‑stator homogenizers are only useful to disrupt soft plants in the presence of lysis
buffer. To minimize foaming, keep the homogenizer submerged at all times.
7.3 LYSIS OF PLANT SAMPLES
Increasing the amount of starting material
The standard protocols of the ISOLATE II Plant DNA Kit facilitate processing of 10‑1500 mg
plant material. This typically yields 1‑300 μg of high quality DNA. However, the amount of
DNA that can be expected per mg of sample depends on both the size and genomic status
(DNA ploidy). For example, 100 mg fresh wheat containing a hexaploid genome (1.7 x 10
10
bp)
contains 30 μg DNA, whereas the same amount of Arabidopsis with a smaller diploid genome
(1.9 x 10
8
bp) only yields 3 μg DNA.
To obtain a sufficient DNA yield, it may therefore, be advantageous to process a higher than
recommended starting mass (up to 5‑fold). However, to ensure complete lysis, all lysis buffer
volumes (protocol step 2) must be increased proportionally and require multiple loading
steps.
Selecting the optimal lysis buffer
Heterogeneity in structures of wall polysaccharides, polyphenols and other components in
plants, can result in suboptimal DNA extraction or performance in downstream applications.
Therefore, two different lysis buffers are provided for optimal processing, purification
performance and yield with most common plant species.
The standard protocol uses Lysis Buffer PA1 based on the well‑established CTAB procedure.
Additionally, Lysis Buffer PA2 (SDS‑based) is supplied which requires subsequent protein
precipitation by potassium acetate (Precipitation Buffer PL3). For some plant species, Lysis
Buffers PA1 and PA2 can be used with similar results. For most plant material, however,
the differing lysis efficiency is due to the negative charge of SDS and the positive charge of
CTAB.