Page 15 - BM 2012 vol.3 No.2

Bioscience Methods 2012, Vol.3, No.2, 7-20
http://bm.sophiapublisher.com
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Figure 12 General Tissue culture strategy adopted
in liquid nitrogen through mixture mill. Extraction
buffer (700 μL) were added to each eppendorf tube
and inverted. Detail of extraction buffer is shown in
table 8. Then 800 μL phenol chloroform isomyl
alcohol, (25:24:1) was added into each eppendorf
tube. Centrifugation was performed for 3 minute at
5 000 rpm at 4℃. Supernatant was taken in to each
new eppendorf followed by addition of 1/10 of sodium
acetate. Then equal volume of Iso-propanol was added
into each tube. Centrifugation was done at 13 200 r/min
for 15 minutes. Supernatant was removed. Washing of
pellets with 80% ethanol was done, followed by air
drying the pellets. The pellet was dissolved in R40
(RNase and TE). DNA quality was checked by
running the isolated DNA samples on agarose gel
electrophoresis. DNA quantity was determined by
Table 8 Genomic DNA Extraction buffer
DNA Extraction buffer Content
Lauryl Sarcosyl 1%
Tris HCl 100 mmol/L
NaCl 100 mmol/L
EDTA 10 mmol/L
using nanophotometer.
3.6 RAPD (PCR) analysis
For determining genetic stability of in vitro regenerated
plants of selected combination compared with wild
type, five random primers were used (Table 9). Random
amplified polymorphic DNA (RAPD) analysis of
genomic DNA was carried out in 25 μL reaction
mixture containing 2.5 μL of template (Genomic
DNA), 3 μL MgCl 2, 4 μL of dNTPs, 0.2 μL of Taq
Polymerase and 2 μL of primer in 1×reaction buffer.
The amplification reaction was performed in the
Master cycler with an initial denaturation for 5 minutes
at 94 ℃, followed by 40 cycles: 1 minute denaturation
at 94 ℃; 1 minute annealing at 36℃; 2 minute extension
Table 9 RAPD primers with their sequence and annealing
temperature
Primer name Primer sequence Annealing Temperature
GL Decamer K- 07 AGCGAGCAAG 36℃
GL Decamer K- 20 GTGTCGCGAG 36℃
GL Decamer B- 02 TGATCCCTGG 36℃
GL Decamer B- 03 CATCCCCCTG 36℃
GL Decamer D- 11 AGCGCCATTG 36℃