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Computational Molecular Biology 2014, Vol. 4, No. 12, 1-8
http://cmb.biopublisher.ca
3
(http://flypush.imgen.bcm.tmc.edu/primer/) was used
to find out the overlapping primers.
1.7 In-silico amplification
The designed primers might amplify or not in wet lab
technique, hence the in-silico method was performed
to check the successful amplification. The
Insilico
PCR amplification tool (http://insilico.ehu.es/PCR/)
was used to find out the amplification. Both forward
and reverse primers were uploaded and after
submitting it showed the amplification.
1.8 Genomic DNA isolation
The genomic DNA isolation process was followed by
using CTAB powder method (Murray and Thompson,
1990). The cut pieces of rice leaf variety HEENA and
HGN1 were homogenized using liquid nitrogen. The
fine powder leaves were then kept in -20 degree
centigrade freezer for 12 hours. 10ml of extraction
buffer was added and incubated at 56 degree
centigrade for 20minutes at 150rpm in shaker. 10ml of
chloroform: isogamy alcohol mixture was added to get
an emulsion. Emulsion was transferred to 30ml
Oakridge polypropylene tube and centrifuged at
8000rpm for 25minutes at room temperature. 600ul of
10% CTAB and chloroform: isogamy alcohol (24:1)
was added and subjected to centrifuge for 25minutes
at room temperature. 0.7 volume of cold filter
sterilized iso-propanol was added, precipitated dna
was spooled out and again subjected to centrifuge at
5000-6000rpm/10minutes, rinsed in 70% ethanol, air
dried or vaccum dried and dissolved in 4ml of TE
buffer. The sample was then treated with 4ul of DNA
free rnase and incubated at 37 degree centigrade for 1
hour. The DNA was re-precipitated with 0.1 volume
of 3m sodium acetate and 2 volume of cold absolute
ethanol. The precipitated DNA was spoole, rinsed in
70% ethanol, dissolved in 1ml of TE and stored at -20
degree centigrade for further analysis. Agarose gel
electrophoresis method was done to find out the
quality of the DNA. Gradient PCR was performed by
setting the annealing temperature of 53
o
C, 54
o
C, 56
o
C
and 57
o
C. The PCR amplification was performed in a
20 µl reaction mixture volume containing 2 µl (30 ng)
of genomic DNA, 1×PCR buffer, 200 µM dNTP mix,
4 pM of each of forward and reverse primers, 2 mM of
magnesium chloride, 1 U of
Taq
(
Thermos aquaticus
)
DNA polymerase
.
The reaction mixture (master mix) was prepared as follows:
Reagent
Final concentration Vol. inµl
Sterile de-ionized
water
-
12.04 ×12 = 144.48
10 X PCR buffer 1×
2.0 ×12 =24.00
10mM dNTP mix
200µM of each
0.16 ×12 =1.92
25 mM magnesium
chloride
2m M
0.8 ×12 =6.0
Primer ( F)
4pM
1 ×12 =12.0
Primer( R)
4pM
1 ×12 =12.0
Taq
DNApolymerase 1U
1 ×12 =12.0
18.0 ×12 = 216.0
The reaction mixture was mixed well, 18 µl was
distributed to each of 12 tubes. Two µl of DNA
sample each genotype was added to corresponding
tube, mixed well, briefly centrifuged to collect drops
from wall of tube.
1.9 Gel electrophoresis and detection of amplified
products
After gradient PCR, the products were separated on
2.5- 3% agarose gel. Twelve micro liters of amplified
products of each sample was loaded on 2.5% agarose
gel in 1× TBE buffer to separate the amplified
fragments. The electrophoresis was done for about 3
hours at 70 volts. The molecular weight marker (50 bp
ladder) was used to compare the molecular weights of
amplified products. 14 µl of Ethidium bromide was
added to the molten gel and mixed well before
pouring to the mold. The gel was visualized under UV
and photographed using Chemi-Doc system (Alpha
Innotech).
1.10 Data analysis
DNA fragment sizing and matching was done by
scoring photographs directly. Individual bands within
lanes were assigned to a particular molecular weight
comparing with the DNA molecular weight marker.
The amplified bands/alleles were scored as present (+)
or absent (-) for each genotype and primer
combination.