Volume 04 Issue 04-2022
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The American Journal of Applied sciences
(ISSN
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2689-0992)
VOLUME
04
I
SSUE
04
Pages:
1-6
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I
MPACT
FACTOR
(2020:
5.
276
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5.
634
)
(2022:
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176
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OCLC
–
1121105553
METADATA
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–
7.987
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ABSTRACT
This article provides information on DNA extraction from plant tissues, their methods, and the effective STAB method.
It also provides detailed information on the tools required to implement the STAB method, the reagents, and the
sequence of operations.
KEYWORDS
DNK, plant, genotype, method, tissue, stab, supernatant, sediment, leaf, homogenization.
INTRODUCTION
Methods of DNK extraction from plant cells
The first step in analyzing plant genotypes is to
perform DNK extraction from plants. In doing so,
researchers used a number of methods. Examples
include:
1.
Phenol-chloroform method. This is an old but
useful method. The downside is that the reagents
used are dangerous to human health and do not
always contain quality DNK for PCR and sequins.
2.
STAB method. It is a very useful method for the
plant. In practice, total plant DNA can be isolated.
Research Article
STAB SEPARATION FROM PLANT ISSUE BY STAB
Submission Date:
March 26, 2022,
Accepted Date:
April 02, 2022,
Published Date:
April 16, 2022 |
Crossref doi:
https://doi.org/10.37547/tajas/Volume04Issue04-01
Dilshod Akhmadovich Mullayev
Doctor of Philosophy (PhD), Acting Associate Professor, Department of "Biology and its teaching
methods", Tashkent State Pedagogical University named after Nizami, Uzbekistan
Khusnorahon To'lqin qizi Tojiboyeva
Student, Tashkent State Pedagogical University named after Nizami, Uzbekistan
Journal
Website:
https://theamericanjou
rnals.com/index.php/ta
jas
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
Volume 04 Issue 04-2022
2
The American Journal of Applied sciences
(ISSN
–
2689-0992)
VOLUME
04
I
SSUE
04
Pages:
1-6
SJIF
I
MPACT
FACTOR
(2020:
5.
276
)
(2021:
5.
634
)
(2022:
6.
176
)
OCLC
–
1121105553
METADATA
IF
–
7.987
Publisher:
The USA Journals
On the plus side, reagents used are less dangerous
to human health and cheaper than other methods.
The downside is that it takes a lot of time.
3.
KIT method. In this method, several steps are
shortened by the addition of reagents in
accordance with each other, compared to other
methods. On the plus side, DNK is of good quality
and breaks down very quickly. The downside is that
it is very expensive. [1]
There are a number of difficulties in extracting pure
and high-quality DNA from plants. For example, due to
the high molecular weight compounds present in
plants, a lot of time and reagents are spent on the
separation of nucleic acids [2].
Most plant species contain high molecular weight
polysaccharides, polyphenols, several pigments, and
other secondary metabolites.
It is very important to select the leaf tissue of plants to
isolate DNK. DNK extraction is effective due to the low
content of polysaccharides and polyphenols in young
eagles [3].
There are some ways to reduce the stages of DNK
extraction, but they involve the use of large amounts
of plant tissue and liquid nitrogen [4].
The method used by Doyle and Doyle (1987) to extract
DNK from plants using liquid nitrogen is widely used. In
recent years, various scientists have been working
more effectively to modify this method. The STAB
method, modified by Doyle and Doyle (1990), has been
successfully applied to many plant species [5].
A number of scientists have also made some changes
to the KIT method to increase its effectiveness.
DNK separation STAB method.
In our study, we used the STAB method and its
modification, which are more useful and popular, to
separate genome DNK from a young leaf of cotton.
DNK buffers, their composition, and preparation:
2 x STAB 300ml
100mM Tris pH-8.0 1MTris pH-8.0 30ml
20mM EDTA pH-8.0 0.5M EDTA pH-8.0 12ml
1.4M NaCl NaCl 24.5448 g
2% STAB STAB 6 g
Volume 04 Issue 04-2022
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The American Journal of Applied sciences
(ISSN
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2689-0992)
VOLUME
04
I
SSUE
04
Pages:
1-6
SJIF
I
MPACT
FACTOR
(2020:
5.
276
)
(2021:
5.
634
)
(2022:
6.
176
)
OCLC
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1121105553
METADATA
IF
–
7.987
Publisher:
The USA Journals
Hold in the autoclave for 30 minutes.
10 x STAB (STAB/NaCl) 100ml
0.7M NaCl NaCl 4.1 g
10%STAB STAB 10 g
Hold on to the autoclave for 30 minutes.
STAB presipitation 100мл
50mM Tris pH-8.0 1M Tris pH-8.0 5ml
10mM EDTA pH-8.0 0.5M EDTA pH-8.0 2ml
1% STAB STAB 1 g
Hold on to the autoclave for 30 minutes.
Highly salty TE 200ml
10mM Tris pH-8.0 1M Tris pH-8.0 2ml
0.1mM EDTA pH-8.0 0.5 M EDTA pH-8.0 40mkl
1M NaCl 11.68 g
Hold on to the autoclave for 30 minutes.
RNKaza
10 ml of RNA is added to 1 ml of dH 2O and soaked in 100
0
C water for 10 minutes
- Stored at 20
0
C.
TE 50 ml
10mM Tris 1M Tris pH 8.0 0.5 ml
1mM EDTA 0.5M EDTA pH 8.0 0.1 ml
Volume 04 Issue 04-2022
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The American Journal of Applied sciences
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2689-0992)
VOLUME
04
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Pages:
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5.
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6.
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0.5 M EDTA pH 8
(Hold on to the autoclave for 30 minutes.)
dH2O 700 ml
Na
2
EDTA2H2O 186.1 g
10N Na OH pH=8.0
dH
2
O 1litr
3M NaOAc pH 5.2
(Hold on to the autoclave for 30 minutes.)
dH
2
O 300 ml
NaC
2
H
3
O
2
3H
2
O(NaOAc) 408 g
Acetic acid concentration pH=5.2 + dH
2
O 1 liter
Xloroform/Izoamil
24ml xloroform 1ml izoamil
REQUIRED EQUIPMENT.
The following laboratory instruments and equipment
were used in the dissertation:
1.
Dallas (Pipetman, USA)
2.
Centrafuga (Eppendorf, Germany)
3.
Thermostatic water bath (LKB, Sweden)
4.
Vortex (Genie Scientific Industries, Inc., USA)
5.
Autoblod (Bellco Glass, Inc., USA)
6.
Horizontal
electrophoresis
equipment
(Stratagene, USA)
7.
Thermostatic mixer (Eppendorf, Germany)
8.
Concentrator (Eppendorf, Germany)
9.
Thermostat (Heraeus, Germany)
10.
Microwave oven (Ferette, Italy)
11.
Scale (Sartorius, USA)
12.
Image Documentation Equipment Alpha Imager
3400 (Alpha Innotech Inc., USA)
SEPARATION OF GENOM DNA FROM PLANT ISSUE BY
STAB.
1.
The leaf is frozen in liquid nitrogen and
homogenized.
2.
Fill 650 μl 2xCTAB and place on AUTOBOT for 20
minutes (stirring every 5 minutes).
3.
Add 650 μl of chloroform: isoamyl (24: 1) and mix
vigorously for 4 minutes.
4.
Centrifuge at 5 min / 10,000 rpm and transfer 550
μl from the top of the supernatant to another
solution.
5.
Add 550 μl of chloroform: isoamyl (24: 1) and mix
vortex.
6.
Centrifuge at 5 min / 10,000 rpm and transfer 500
μl from the top of the supernatant to another
solution.
7.
Add 300 μl of High Salt to the precipitate and
vortex for 5 minutes, then keep it at room
temperature for 15 minutes.
8.
Add 250 μl of isopropanol and mix slowly for 2
minutes and refrigerate at -200 ° C.
Volume 04 Issue 04-2022
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The American Journal of Applied sciences
(ISSN
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2689-0992)
VOLUME
04
I
SSUE
04
Pages:
1-6
SJIF
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(2020:
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(2021:
5.
634
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(2022:
6.
176
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OCLC
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METADATA
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–
7.987
Publisher:
The USA Journals
9.
Centrifuge for 15 min / 10,000 rpm and pour
supernatant.
10.
Add 500 μl of 70% alcohol to the precipitate and mix
for 5 minutes.
11.
Centrifuge at 3 rpm / 14,000 rpm and pour in the
alcohol.
12.
500 μl of 70% alcohol is added to the sediment.
13.
Centrifuge for 3 min / 14,000 rpm and pour in the
alcohol.
14.
Sediment is dried in a DNK concentrator for 10-15
minutes.
15.
Vortex 100 μl of TE buffer onto the sink and place
in a short centrifuge and -200 C refrigerator.
EXAMINATION
OF
DNA
SAMPLES
BY
ELECTROPHORESIS.
DNK electrophoresis in agarose gel is a standard
method used for the purification and identification of
DNK fragments.
The method of electrophoresis of DNK on horizontal
agarose gel plates is widely used in molecular genetics
and biochemistry. Because the reagents of this method
are easy to find, the simplicity of the method and the
low cost of the equipment make it possible to obtain
sufficient information from very small amounts of
untreated material.
Therefore, the experiments use the method of
electrophoresis on horizontal agarose gel plates.
Figure 1 DNK isolated using the Stab method.
Plant DNK samples isolated using the standard STAB
method of DNK separation were electrophoresed in
09% agarose gel. The electrophoresis gel was stained
using Ethidium Bromide and photographed on a UV
Transilluminator (Innotech Inc., USA) (Figure 1).
REFERENCES
1.
Shermatov Sh. E. “biologik ashyolardan DNK
ajratish usullari” // Genom va bioinfarmatika markaz
o’quv matreali // Toshkent. 2009 yil 138-141 bet.
Volume 04 Issue 04-2022
6
The American Journal of Applied sciences
(ISSN
–
2689-0992)
VOLUME
04
I
SSUE
04
Pages:
1-6
SJIF
I
MPACT
FACTOR
(2020:
5.
276
)
(2021:
5.
634
)
(2022:
6.
176
)
OCLC
–
1121105553
METADATA
IF
–
7.987
Publisher:
The USA Journals
2.
Ali Dehestani, S.K. Kazemi Tabar A Rapid Efficient
Method for DNA Isolation from Plants with High
Levels of Secondary Metabolites // Asian Journal of
Plant Sciences // Asian Journal of Plant Sciences,
June 2007, DOI:10.3923/ajps.2007.977.981.
3.
Jinfa Zhang, James Mcd, Stewart Economical and
rapid method for extracting cotton genomic DNA //
Journal of Cotton Science// 4(3):193-201 January
2000.
4.
Raul Tapia-Tussell, Andres Quijano-Ramayo, Rojas
Herrera Rafael, Alfonso Larqué-Saavedra A Fast,
Simple, and Reliable High-Yielding Method for DNA
Extraction From Different Plant Species // Molecular
Biotechnology // November 2005 31(2): 137-9,
DOI:10.1385/MB:31:2:137.
5.
Doyle, J.J.; Doyle J.L. Isolation of plant DNA from
fresh tissue. Focus, v.12, p.13-15, 1990.
