Materials and Methods
Plant Materials
Plant materials for sequencing of ITS sequences of nuclear riboso-
mal DNA were collected during field trip or from the herbarium
specimens. Voucher specimens for the plant material used in the
experiment are listed in [Table-1]. Leaves were dried in silica gel
prior to DNA extraction.
Table 1- Voucher information and GenBank accession number for
the taxon sequenced in the present study
Total Genomic DNA Extraction
Total genomic DNA was extracted using the DNeasy Plant Mini kit
(QIAGEN Inc., Crawley, West Sussex, UK). In brief, 20mg silica gel
dried leaf tissue was taken in 1.5 ml eppendorf cup, and 5-6 tung-
sten carbide bead placed in eppendorf cup. The eppendorf cup
(with leaf and tungsten carbide bead) placed in tissue lyser and
grinded the leaves sample for 2-3 minutes. Eppendorf cup were
taken out from tissue lyser after grinding, and 4 ul RNase stock
solution (100 mg/ml) and 400ul Buffer AP1 was added and vortex.
Buffer AP1 is the main extraction buffer which isolates DNA from
the cells. The purpose of adding RNase (an enzyme) was to break
the RNA into small pieces which remain in the cell, so that easy to
pass from filter. After adding 4 ul RNase and 400 ul Buffer AP1 in to
the eppendorf cup containing grinded leaf tissue, the eppendorf cup
were place in the water bath or dry bath to incubate the mixture of 4
ul RNase, 400 ul Buffer AP1 and grinded leaf tissue for 10 minutes
at 65ºC. It was mixed 2 or 3 times during the incubation period by
inverting the tubes gently. After 10 minutes of incubation the eppen-
dorf cup was taken out from water bath or dry bath, and 130 ul Buff-
er AP2 was added to the eppendorf cup containing Lysate and then
mixed it properly by inverting the tubes and then incubated the ep-
pendorf tube (containing mixture of 4 ul RNase, 400 ul Buffer AP1,
grinded leaf tissue and Buffer AP2) for 5 minutes on ice. After cold
incubation for 5 minute on ice, the eppendorf cup (containing mix-
ture of 4 ul RNase, 400 ul Buffer AP1, grinded leaf tissue and Buffer
AP2) were taken out and kept on the room temperature for 10
minutes. Now the eppendorf cup (containing cold incubated mixture
of 4 ul RNase, 400 ul Buffer AP1, grinded leaf tissue and Buffer
AP2) placed into centrifuge machine and centrifuge the eppendorf
tube (containing cold incubated mixture of 4 ul RNase, 400 ul Buffer
AP1, grinded leaf tissue and Buffer AP2) at 14000 rpm for 5
minutes. After centrifugation, the clear or colorless Lysate
(supernatant, which contains DNA) transferred to QIAshredder mini
spin column lilac- pink in color which is sitting or placed in a 2 ml
collection tube), and centrifuge it for 2 minutes at 14000 rpm. The
clear lysate (obtained into 2 ml collection tube after centrifuge trans-
ferred to the collection tube, and 1.5 volume Buffer AP3/E added to
the clear lysate and mix it by pippeting. AP3/E is the binding buffer.
Now 650ul of the mixture (mixture of clear lysate containing DNA
and buffer AP3/E) was transferred into DNeasy mini spin colorless
column (which are colorless and placed or sitting into 2 ml collection
tube, supplied along with kit) and Centrifuge it for 1 minute at 8000
rpm. The flow through comes out into the collection tube after cen-
trifuge was of no use therefore it was discarded. The process was
repeated with the remaining lysate to get DNA on the filter. The filter
was kept on room temperature before washing. For washing 500 ml
Buffer AW was added to DNeasy mini spin column containing DNA
and centrifuge 2 minutes at 14000 rpm. Flow through was discard-
ed and the process of washing repeated again. After completion of
washing, the DNeasy mini spin column containing DNA was kept on
room temperature for 5 minutes, and then for elution, 100ul steri-
lized double distilled water (pyrogen free) was added to DNeasy
mini spin column containing DNA and centrifuge for 2 minute at
14000 rpm. After elution, the DNA transferred to fresh 1.5 ml ep-
pendorf tube and stored at -20 for further use of PCR.
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