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3.2.3.5.3.25. Molecular analyses of putative transformants
3.2.3.5.3.2.5.1. Genomic DNA extraction
Genomic DNA was extracted following the method described by Dellnporta
cr
al.,
(1983). One gram of leaf of putative transformants of tobacco was frozen in liquid
nitrogcn and ground to a fine powder using pre-chilled pestle and monar. Ground
material was transferred to 50 ml centrifuge tube containing 15 ml of grinding buffer
(100 mM Tris HCI pH 8.0. 50 mM EDTA pH 8.0 and 500 mM NaCI). The contents
were mixed well by gentle inversion and 800 p1 of 2070 SDS was added into that
followed by vigorous mixing. The tubes were incubated at
65
O C for 30 min. After
incubation, 5
m l
of 5 M potassium acetate was added, mixed thoroughly and kept on
ice for 15 min. To this mixture. 12 ml of chloroform:isoamyl alcohol (24:l) was
added, mixed thoroughly and kept on ice for 15 min. The content was centrifuged at
10,000 g for 10 min. at 4 "C. The aqueous phase was transferred to a new
mjcrocentrifuge tube and mixed with equal volume of ice cold isopropanol and kept at
-
20 "C for 20-30 min. After 30 min.. tubes were centrifuged at 10,000 g for LO min.
at 4 "C. The supernatant was discarded, pellet was dried for 5 min. at room
temperature (28
O C )
and dissolved in 400 p1 of sterile water. Then the DNA mixture
was transferred to 1.5
ml
eppendorf tubes and mixed with equal volume of
chloroform:isoamyl alcohol (24: 1). Aqueous phase was transferred to new eppendorf
tubes and mixed with 1/10 volume of 3
M
sodium acetate and 2 volume of 99.9%
(vlv) ethyl alcohol and kept at -20 "C overnight. The DNA was pelleted by
centrifugation at 10,000 g for 15 min. and washed with 70% ethanol before the final
centrifugation. DNA pellet was air dried and dissolved in 100
p 1
of 0.1 X TE buffer.
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