Thermo Scientific KingFisher Plant DNA Kit, Инструкция по эксплуатации

Страница: 22 / 40

Thermo Fisher Scientific 22 Thermo Scientific KingFisher Plant DNA Kit Instruction Manual
Protocols and Pipetting Instructions
Homogenization of sample material
Using young plant samples, and/or if possible keeping
plants for 12 hours in darkness before collecting the
samples, reduces the polysaccharide and polyphenolic
contents, which may interfere in downstream applications.
Plant samples can be stored in frozen or lyophilized form,
or in ethanol.
Efficient homogenization of the sample material is an
essential step before DNA purification in order to gain
a good yield of high-quality DNA. Plant tissue can be
homogenized, for example, with a pestle, using steel
beads or with commercial homogenizers, of which
high-throughput homogenizers offer a suitable method for
handling 96 samples simultaneously. The homogenization
step must disrupt the structures of the starting material
rapidly and completely in order to ensure a high yield of
DNA.
Homogenize 20–50 mg of fresh plant sample (or less if
dried samples are used). After homogenization, add 500 µl
of Lysis Buffer to a homogenized sample and mix for
30 seconds. If RNase A treatment is needed to reduce the
amount of RNA in the sample, add RNase A to the Lysis
Buffer at a final concentration of 0.25 mg/ml. RNase A
treatment is recommended for the samples containing
large amounts of RNA. Centrifuge the sample briefly in
the Lysis Buffer (30 seconds, 1500 x g) and incubate at
56°C for 30 minutes.
To clear the plant lysate, centrifuge the samples for
20 minutes (6000 x g). Transfer the supernatant to a
Thermo Scientific Microtiter deep well 96 plate and begin
the purification of DNA using the KingFisher Flex or
KingFisher Duo. See the detailed instructions below.
Homogenization of
sample material