Optimization of DNA extraction from seeds and fresh leaf tissues of Soybean prosiding
PROCEEDING OF
INTERNATIONAL BIOLOGY CONFERENCE
Science for Energy, Food and Environmental Sustainability
December 6th, 2012
Seminar Room, SCC (Student Community Center) Building
lnstitut Teknologi Sepuluh Nopember
Sukolilo ｾ＠ Surabaya
IBOC2012
- -
.........
Wli!iiiiH!'f!tilllll!4€4
Ｍ
Ｍｾ
ﾷＮ＠
INTERNATIONAL BIOLOGY CONFERENCE
Organized by
Department of Biology, lnstitut Teknologi Sepuluh Nopember (ITS}, Surabaya -Indonesia
Supported by
lnstitut Teknologi Sepuluh Nopember (ITS), Surabaya- Indonesia
iTS
lnstitut
Teknologi
Sepuluh Nopember
Proceeding of International Biology Conference (IBOC) 2012: Science for Energy, Food and
Environmental Sustainability
© Department of Biology, Faculty of Mathematic and Natural Sciences, lnstitut Te knologi Sepu luh
Nopember- Surab aya, Indonesia
Text and layout editor
Farid K. Muza ki, S.Si, M .Si
Roksun Nasihin, S.S i
Talitha Rahma Nathan ia, S.Si
Aplilia Fitriana, S.Si
Dinda Zahidah, S.Si
Andarini Ayu R, S.Si
Front cove r
lwenda Bella Subagio, S.Si
All rights reserve d. No part of this manuscript may be reproduced, stored in a retrieval system, or
tran smitted by an y other means without the prior perm ission of the copyright holder. Plea se direct
al l enquiries to th e publishers .
i IS BN 978 -97 9-9 731 6-1 -6
I
Depa rtm ent of Biology
Faculty of Mathematic and Natura l Sciences
lnst itut Tekno log i Sepu luh Nopember
Kam pus ITS Sukoli lo
Jl. A. R. Haki m, Kep utih- Su koli lo, Surabaya 6011 1
www.bi o .it s.ac. id
PREFACE
We would like to thank to God for bless ing us until we are able to organize our first international
annual event called The International Biology Conference (IBoC) 2012 at the end of this year. This
conference is purposed to build a future and promising international networking between our
department and other international parties which have a similar interest keeping environment
balance for save the earth . Concerning to it, our first theme is "Science for Energy, Food and
Environmental Su stai nability" .
The first outcome we would gain is having a direct and personal contact, sharing and discussion with
sc ientists around the world . Further on we would like to build a real scientific networking doing a real
w ork keeping the earth balance and save . We are really grateful realizing that they are from tgypt,
Thailand, Bangladesh, Malaysia and Indonesia . Thank you for your participations .
We are also glad to inform that, this conference have been possible only because of the support from
The Faculty of Mathematics and Natural Sciences and The lnstitut Teknologi Sepuluh Nopember (ITS),
Surabaya-lndonesia. The Vice Rector, Prof. Darminto put several administrative and scientific advices.
The big applause and thank is also going to our students from The Biology Department who has been
managing the conference .
Thank you and best regards.
Surabaya, June 51h 2013
Head of Biology Department
Dr.rer.nat. Maya Shovitri
CONTENTS
Preface
Contents
Utilization of both cellulose and hemicellulose in agricultural solid waste for
production of biofuel- Arief WIDJAJA
1
Purification and characterization of autoinducer-2 inactivating enzyme in
periodontopathogenic bacterium Eikenella corrodens- Fariha Jasin MANSUR, Mohammad
Minnatul KARIM, Ayako NAGAO and Hiroyuki AZAKAMI
3
Effect of Autoinduce r-2 on biofilm formation of periodontopathogenic bacterium
5
Eikenella corrodens - Mohammad Minnatul KARIM, Tatsunori HISAMOTO, Yuichiro NOIRI,
Shigeyuki EBISU, and Hiroyuki AZAKAMI
Initial study on fisheries resources in Lamong estuary, East Java
- Farid K. MUZAK!, Apri liana Mutia DEWI and D. Ali SAUWIBI
7
The effectivity of Micorhiza against Fusarium withered disease
in Tomato (Lycopersicon esculentum Mill.} Fortuna variety- YUNIS, Sri NURHATIKA and
Krintansi lndah PURWANI
17
Effect of Rhizobium and indigenous Mycorrhiza from Labang, Bangkalan- Madura
on the growth of peanut (Arachis hypogea)- Tutik NURHIDAYATI, Kristanti lndah PURWANI,
Ni Ketut Dewi INDRAYANI and Triano Bagus SAPUTRO
31
Feed conversion of Silver Pompano (Trachinotus blochii) in culture media using
manipulated salinity- Nurlita ABDULGANI, Salim ARROKHMAN and Dewi HIDAYATI
35
Biosurfactant production by Pseudomonas putida T1 (8} using molasses as substrate
- Nur Hidayatul ALAMI, Lailatul MAGHFIROH, FATIMAH and NI'MATUZAHROH
41
Isolation and identification of Acetobacter from banana peels native Indonesia as a
potential bacteria for banana vinegar production - Dadang H.MUSTHOFA, Jonathan
lvander KURNIAWAN Nurlaila SHOVIANITA, Ahmad ARIFIYANTO, Roksun NASIKHIN and Maya
SHOVITRI
49
Hydrogen gas produced by isolated bacteria from septic tank- Maya SHOVITRI,
engah D. Kuswytasari, Ayuk RAHMAWATI and Elita S. AMBARNINGTYAS
59
Production of cellulase enzymes by Penici/lium sp in temperature, pH and different
agricultural waste- Irma ALFIAH and Nengah Dwianita KUSWYTASARI
67
Bi rd diversity in different types of wetland area in Keputih,
East Coast of Surabaya - Hubertus B. AJIE and Citra Fitrie RIANY
75
istribution study of birds conserved by Indonesian law in Wonorejo, Surabaya
- s a DESMAWATI, AUNUROHIM and lndah TRISNAWATI
83
ｾＺＮ＠
ｾ＠
--ｾ＠ :ＭｾＬ
ｴＺｾ［ＭﾷＮｳ＿｟Ｌ＠
.
r-
.
.-
ｾ＠
ＺＮｾＬ＠
.
,.•_
［ｾＧ＠
Study on habitat and bird community at western Mbeliling landscape, Flores
95
- Nur Sit a HAMZAT I, Fer i IRAWAN , AUNUROHIM
Nuclear markers in biodiversity studies of some black corals species (Antipatha ri a,
Hexacorallia) of North Sulawesi, Indonesia- Hapry F.N LAPIAN
103
Status of coral reefs before and after mass bleaching event 2010 in coastal water of
PLTU Paiton - Farid K. MUZAKI and Dian SAPTARINI
111
Exploration of potential bacteria as biofertilizer from soil samples of Alas Purwo
National Park - Agu s SUPRIYANTO, Tini SURTININGSIH, Tri NURHARIYATI, NI' MATUZAH ROH
119
Bioremediation of cooking oil contaminated soil using hydrocarbonoclactic
microbial consortium isolated from Wonocolo- Bojonegoro- NI' MATUZAHROH , ln dana
125
ZULFA, Nur Hidayatul ALAMI, Tri NURHARIYATI
Grouping of epiphytes meiofauna on seagrass using canonical correspondence
analysis - lndah TRISNAWATI, M . MURYONO, M . Sjahid AKBAR, KURNIAWAN
133
Optimization of DNA extraction from seeds and fresh leaf tissues
of Soybean (Glycine max)- Oeke YUNITA, Benny SETIAWAN and EVELYN
141
Potential Arecaceae family for bioenergy, food, medicine
147
and environmental services in Purwodadi Botanic Garden- Rony IRAWANTO
Bacteria biodiversity in cow dung, capsule husk and seed cake
of Jatropha curcas Linn. anaerobic digesters - Roy HENDROKO, Ahmad WAHYUDI, Elizabeth
155
C. SITU MORANG, Nurita TORUAN and Satriyo K. WAHONO
Influence of incubation temperature and pH on activity of ligninolytic enzymes
in sugarcane baggase by Gliomastix sp. T3.7 - lma MufidatuiiLMI, Nengah Dwia nita
161
KUSWYT ASA RI
The hypnotic effect of benzoylthioutea derivative {2.4 dichlorobenzoylthiourea)
in mice - Di ni KESUMA
167
The motor coordination disorder effect of 2-chlorobenzoylthiourea in mice - Agusli na
175
KIRTISH ANTI
Community structure of benthic fauna in rocky shores of Balekambang - lwe nda Bella
183
SUBAGI O, Di mas Ca hyo HARTRANTO and Erna ROFIDAH
The utilizat ion of potential Durian peel waste namely Biobriquettes as energy
resource - Wahidin NURIANA, Nurfa ANISA and MARTANA
189
Measured and modeled plant growth and productivity in relation to SRI (System of
Rice Intensification) method on local paddy cultivar under salinity stress - Mu khammad
191
MURYONO and lndah TRISNAWATI
Diversity and speci es distribution of mangrove in East Java - Fari d K. MUZAKI, Dian
SAPTARIN I, Syaik hul MAHM UD, Muham mad ROM ADH ON I, Lind a Novit a SARI and Sofyan ARIS
193
Fun ctional biod iversity of Arthropods fa un a following seasonal variation in the
195
agri cultural landsca pe - lndah TRI SNAWATI and Mukhammad MURYONO
Study of Potential Phytoplankton as a Cause of HABs (Harmful Algal Blooms) in
197
Northern Coast of East Java- Dian SAPTARIN I, Fit ri Wu land ari EFFENDI, Wenny D. D.
RA HM ADIANI and Citra Fitri e RIANY
Toxi city test and ch e mical screening of Term inalia catappa leaf extract as a
199
bi ofungicide to control Phytopthora capsid growth on red pepper (Capsicum
frutescens longa .) - Dini ERMAVITALIN I, Kristan ti lndah PU RWANI , Tuti k NURHIDAYATI, Sri
URHATI KA
Optimisation of biosurfactant oroduction from Acinetobacter sp. P2(1) using glucose
201
substrate- FATIMAH, SUHARJONO, NI' MATUZAHROH, Tri ARDYATI, Afaf BAKTIR, Ahmad
HON TOW I
Characterize lypolitic and proteolytic bacteria from domestic sewage - Nita CITRASARI,
I' MATUZAHROH and Tri NURHARIYANTI
203
Isolation of HS Avia n Influenza viruses from domestic poultry in Surabaya, East Java,
205
In donesia, in 2010 - 2011 - Luh Ade Wilan KRISNA, Masaoki YAMAOKA, Aldise Mareta
ASTRI, Emmanuel Djoko POETRANTO, Laksmi WULANDARI , Resti YUDHAWATI, Landia
SETIAWATI, Retno Asih SETYONINGRUM, Teridah Ermala GINTING, Akiko MAKINO, Kyoko
_HI NYA and Yoshihiro KAWAOKA
Effectiveness of Moringa o/eifera seeds to reduce levels of Pb and Cr in Surabaya River
207
- AU NU ROHIM, Nenga h Dwianita KUSWYTASARI and MUHARTO
Opi sthobranchia of East Java- Fa rid Kama l MUZAK!, Dian SAPTARINI and Swiss WINNASIS
209
e composition of i nsects in farming land of ITS Campus- Surabaya- NURYATI, lndah
---:> JSN AWATI, Kristanti ln dah PURWANI
211
Bird diversity and status in Lamong Bay Estuary, Surabaya- Ahmad YANUAR, Febri Eka
213
］ｾｄ
ａｎ
ｾｰｬｩ
｣ ｡ｴｩｯｮ＠
ａ＠
indegenus Vessicula Arbuscular Mycorrhiza (VAM) from Socah village,
. 215
Ban gkalan-Madura a nd Rhizobium of Peanut (Arachis hypogea)- Tutik NURH IDAYATI
::-o Tri an o Bagus SAPUTRO
;J
eli m inary study of Sidoarjo mud effluent effect to the water quality and fish survival
217
-;3t e - Dewi HIDAYATI, Norela SULAIMAN, M .Shuhaimi -OTHMAN and Ismail B.S
Esti m ation of carbon stock on Mangrove in Pamurbaya - Nur jannatul FAIDAH,
bi hatuz ZAHRO H, Sofyan ARIS, Putri LILIAN DAR!, Vita KARLINDA
219
Optimization of DNA extraction from seeds and fresh leaf tissues of Soybean (Glycine max)
Oeke YUNITA, Benny SETIAWAN and EVELYN
Faculty of Pharmacy
University of Surabaya
Ema il: oeke .ynita@gmail.com
Abstract
The effects of various components on extraction buffer such as SDS-NaCI, PVP, ｾ Ｍ ｭ･ｲ｣｡ｰｴｯｨ
ｮ ｯｬ
Ｌ＠
extraction with Phenoi:Chloroform :lsoamyl acetate, and incubation time on the DNA extraction from
seeds and fresh leaves of Soybean (Glycine max) were stud ied. Based on results above, an optimized
method for DNA extraction from Soybean seeds and leaves were established. Extracting Soybean
seeds twice with Phenoi :Chloroform:lsoamyl acetate (25:24:1), incubating for 30 min and 1% SDS-2
M NaCI in extraction solution could promote the quantity and purity of DNA from seeds, respect ive ly.
The results also showed th at high quality DNA from Soybean leaves could be extracted with
Phenoi:Chloroform:lsoamyl acetate (25:24:1) twice, incubating for 30 min and 0.5% SDS-2% PVP in
extraction solution, although the DNA quantity was less than DNA quantity obtained with
Nucleospin· Plant II method .
Keywords
:Glycine max, DNA extraction, optimization
INTRODUCTION
Soybean (Glycine max) is an important commodity in Indonesian Protective Commitment in
International Trading Confe rence (WT0)( 1 ) which is used in food and herbal supplement.(2)
Despite its important effect as herbal supplement, there were many reports that revealed its side
effects on skin, gastrointestinal, and respiratory reactions and in some cases anaphylaxisYl Before
further evaluation about its side effect, correct identification of the raw material must be performed
to ensure its safety. One of the most reliable methods for identification of herbal medicine materials
is by analyzing DNA. DNA markers are reliable for informative polymorphisms as the genetic
composition is unique for each species and is not affected by age, physiological conditions as well as
environmental factors. (4 )
The application of genetic identification in some plant species has however been constrained by lack
of efficient DNA extraction techniques, because of the presence of polyphenols and metabolites that
interfere with further application of DNA, such as DNA fingerprinting. (S) Therefore this study had
performed optimization of DNA extraction from seeds and fresh leaves of soybean .
MATERIAL AND METHODS
Plant material
Soybean seeds were collected from local market at Surabaya on 2011. After being cultivated several
days in the soil, the leaves from the seeds were collected, washed free of dirt, mopped dry and
quickly stored at -80 2C until used.
Solutions
The extraction buffer cons isted of 200 mM Tris-HCI pH 8.0, 250 mM NaCI, 25 mM EDTA, and 0.5 _
SDS was prepared. The following solutions were also prepared and stored
at
4•c
Phenoi:Chloroform: lsoamylacetate (25:24:1), 70% Ethanol.
DNA extraction
6
Genom ic DNA extraction, modified from ICI Seeds Co.1996( l. Seeds (Fresh leaves) 100.0 mg we re
ground and ＴＰｾＭｴｬ＠
of extraction buffer (200 mM Tris-HCI pH 8.0, 250 mM NaCI, 25 mM EDTA, anc
0.5% SDS) were added . The mixture was added w ith ＴＰｾＭｴｬ＠
phenol with occasi onal inversion and th er
centrifuged 12,000rpm 4•c for 10 min. Three hundreds micro littre of upper phase was mixed wi-ＲＵＰｾＭｴｬ＠
isopropanol and incubated at room temperature for 10 min. After centrifugation 12,000 g c: :
42C for 10m in, the DNA pellet was washed with
ＶＰｾＭｴｬ＠
4•c for 10 min and DNA pe llet was dissolved in ＳＰｾＭｴｬ＠
of 70% cold ethanol, centrifuged 12,000rpr
sterile water (for leaves) or ＱＰｾＭｴｬ＠
(for see s
and kept at -20 2( until used.
Optimization of DNA extraction from Soybean seeds and leaves
Based on extraction procedure above, following modifications on DNA extraction method
done
respectively:
(1)
with 2% PVP for leaves;
ｷ
･
ｾ＠
(2) DNA extraction with phenol, Phe nc
Ch loroform:lsoamyl acetate (25 :24:1) once and tw ice; (3) set a different incubation time (0 mi n,
3:
min, and 60 min); and (4) adjust the concentration of SDS in extraction solution to 0.5%, 1.0% a :
2.0%, respectively; (5) adjust the composition of SDS and NaCI (0.5% SDS- 250mM NaCI; 1% SDS-2'
NaCI) f o r seeds; (6) adjust th e concentration of 6-mercaptoethano/ (1.0%, 5.0%, and 10.0%) fc·
leaves.
Detection of quality and quantity of DNA from the seeds and leaves of Soybean
DNA
purity
(A260/A280
ratio)
and
its
concentration
were
measu red
with
Nano Drc:
spectrophotometer.
RESULTS and DISCUSSION
This study had used seeds and leaves of soybean fo r extracting the DNA. Soybean seeds were useo this study because seeds frequently being sold at the local market and they were usually cons ulT'
INTERNATIONAL BIOLOGY CONFERENCE
Science for Energy, Food and Environmental Sustainability
December 6th, 2012
Seminar Room, SCC (Student Community Center) Building
lnstitut Teknologi Sepuluh Nopember
Sukolilo ｾ＠ Surabaya
IBOC2012
- -
.........
Wli!iiiiH!'f!tilllll!4€4
Ｍ
Ｍｾ
ﾷＮ＠
INTERNATIONAL BIOLOGY CONFERENCE
Organized by
Department of Biology, lnstitut Teknologi Sepuluh Nopember (ITS}, Surabaya -Indonesia
Supported by
lnstitut Teknologi Sepuluh Nopember (ITS), Surabaya- Indonesia
iTS
lnstitut
Teknologi
Sepuluh Nopember
Proceeding of International Biology Conference (IBOC) 2012: Science for Energy, Food and
Environmental Sustainability
© Department of Biology, Faculty of Mathematic and Natural Sciences, lnstitut Te knologi Sepu luh
Nopember- Surab aya, Indonesia
Text and layout editor
Farid K. Muza ki, S.Si, M .Si
Roksun Nasihin, S.S i
Talitha Rahma Nathan ia, S.Si
Aplilia Fitriana, S.Si
Dinda Zahidah, S.Si
Andarini Ayu R, S.Si
Front cove r
lwenda Bella Subagio, S.Si
All rights reserve d. No part of this manuscript may be reproduced, stored in a retrieval system, or
tran smitted by an y other means without the prior perm ission of the copyright holder. Plea se direct
al l enquiries to th e publishers .
i IS BN 978 -97 9-9 731 6-1 -6
I
Depa rtm ent of Biology
Faculty of Mathematic and Natura l Sciences
lnst itut Tekno log i Sepu luh Nopember
Kam pus ITS Sukoli lo
Jl. A. R. Haki m, Kep utih- Su koli lo, Surabaya 6011 1
www.bi o .it s.ac. id
PREFACE
We would like to thank to God for bless ing us until we are able to organize our first international
annual event called The International Biology Conference (IBoC) 2012 at the end of this year. This
conference is purposed to build a future and promising international networking between our
department and other international parties which have a similar interest keeping environment
balance for save the earth . Concerning to it, our first theme is "Science for Energy, Food and
Environmental Su stai nability" .
The first outcome we would gain is having a direct and personal contact, sharing and discussion with
sc ientists around the world . Further on we would like to build a real scientific networking doing a real
w ork keeping the earth balance and save . We are really grateful realizing that they are from tgypt,
Thailand, Bangladesh, Malaysia and Indonesia . Thank you for your participations .
We are also glad to inform that, this conference have been possible only because of the support from
The Faculty of Mathematics and Natural Sciences and The lnstitut Teknologi Sepuluh Nopember (ITS),
Surabaya-lndonesia. The Vice Rector, Prof. Darminto put several administrative and scientific advices.
The big applause and thank is also going to our students from The Biology Department who has been
managing the conference .
Thank you and best regards.
Surabaya, June 51h 2013
Head of Biology Department
Dr.rer.nat. Maya Shovitri
CONTENTS
Preface
Contents
Utilization of both cellulose and hemicellulose in agricultural solid waste for
production of biofuel- Arief WIDJAJA
1
Purification and characterization of autoinducer-2 inactivating enzyme in
periodontopathogenic bacterium Eikenella corrodens- Fariha Jasin MANSUR, Mohammad
Minnatul KARIM, Ayako NAGAO and Hiroyuki AZAKAMI
3
Effect of Autoinduce r-2 on biofilm formation of periodontopathogenic bacterium
5
Eikenella corrodens - Mohammad Minnatul KARIM, Tatsunori HISAMOTO, Yuichiro NOIRI,
Shigeyuki EBISU, and Hiroyuki AZAKAMI
Initial study on fisheries resources in Lamong estuary, East Java
- Farid K. MUZAK!, Apri liana Mutia DEWI and D. Ali SAUWIBI
7
The effectivity of Micorhiza against Fusarium withered disease
in Tomato (Lycopersicon esculentum Mill.} Fortuna variety- YUNIS, Sri NURHATIKA and
Krintansi lndah PURWANI
17
Effect of Rhizobium and indigenous Mycorrhiza from Labang, Bangkalan- Madura
on the growth of peanut (Arachis hypogea)- Tutik NURHIDAYATI, Kristanti lndah PURWANI,
Ni Ketut Dewi INDRAYANI and Triano Bagus SAPUTRO
31
Feed conversion of Silver Pompano (Trachinotus blochii) in culture media using
manipulated salinity- Nurlita ABDULGANI, Salim ARROKHMAN and Dewi HIDAYATI
35
Biosurfactant production by Pseudomonas putida T1 (8} using molasses as substrate
- Nur Hidayatul ALAMI, Lailatul MAGHFIROH, FATIMAH and NI'MATUZAHROH
41
Isolation and identification of Acetobacter from banana peels native Indonesia as a
potential bacteria for banana vinegar production - Dadang H.MUSTHOFA, Jonathan
lvander KURNIAWAN Nurlaila SHOVIANITA, Ahmad ARIFIYANTO, Roksun NASIKHIN and Maya
SHOVITRI
49
Hydrogen gas produced by isolated bacteria from septic tank- Maya SHOVITRI,
engah D. Kuswytasari, Ayuk RAHMAWATI and Elita S. AMBARNINGTYAS
59
Production of cellulase enzymes by Penici/lium sp in temperature, pH and different
agricultural waste- Irma ALFIAH and Nengah Dwianita KUSWYTASARI
67
Bi rd diversity in different types of wetland area in Keputih,
East Coast of Surabaya - Hubertus B. AJIE and Citra Fitrie RIANY
75
istribution study of birds conserved by Indonesian law in Wonorejo, Surabaya
- s a DESMAWATI, AUNUROHIM and lndah TRISNAWATI
83
ｾＺＮ＠
ｾ＠
--ｾ＠ :ＭｾＬ
ｴＺｾ［ＭﾷＮｳ＿｟Ｌ＠
.
r-
.
.-
ｾ＠
ＺＮｾＬ＠
.
,.•_
［ｾＧ＠
Study on habitat and bird community at western Mbeliling landscape, Flores
95
- Nur Sit a HAMZAT I, Fer i IRAWAN , AUNUROHIM
Nuclear markers in biodiversity studies of some black corals species (Antipatha ri a,
Hexacorallia) of North Sulawesi, Indonesia- Hapry F.N LAPIAN
103
Status of coral reefs before and after mass bleaching event 2010 in coastal water of
PLTU Paiton - Farid K. MUZAKI and Dian SAPTARINI
111
Exploration of potential bacteria as biofertilizer from soil samples of Alas Purwo
National Park - Agu s SUPRIYANTO, Tini SURTININGSIH, Tri NURHARIYATI, NI' MATUZAH ROH
119
Bioremediation of cooking oil contaminated soil using hydrocarbonoclactic
microbial consortium isolated from Wonocolo- Bojonegoro- NI' MATUZAHROH , ln dana
125
ZULFA, Nur Hidayatul ALAMI, Tri NURHARIYATI
Grouping of epiphytes meiofauna on seagrass using canonical correspondence
analysis - lndah TRISNAWATI, M . MURYONO, M . Sjahid AKBAR, KURNIAWAN
133
Optimization of DNA extraction from seeds and fresh leaf tissues
of Soybean (Glycine max)- Oeke YUNITA, Benny SETIAWAN and EVELYN
141
Potential Arecaceae family for bioenergy, food, medicine
147
and environmental services in Purwodadi Botanic Garden- Rony IRAWANTO
Bacteria biodiversity in cow dung, capsule husk and seed cake
of Jatropha curcas Linn. anaerobic digesters - Roy HENDROKO, Ahmad WAHYUDI, Elizabeth
155
C. SITU MORANG, Nurita TORUAN and Satriyo K. WAHONO
Influence of incubation temperature and pH on activity of ligninolytic enzymes
in sugarcane baggase by Gliomastix sp. T3.7 - lma MufidatuiiLMI, Nengah Dwia nita
161
KUSWYT ASA RI
The hypnotic effect of benzoylthioutea derivative {2.4 dichlorobenzoylthiourea)
in mice - Di ni KESUMA
167
The motor coordination disorder effect of 2-chlorobenzoylthiourea in mice - Agusli na
175
KIRTISH ANTI
Community structure of benthic fauna in rocky shores of Balekambang - lwe nda Bella
183
SUBAGI O, Di mas Ca hyo HARTRANTO and Erna ROFIDAH
The utilizat ion of potential Durian peel waste namely Biobriquettes as energy
resource - Wahidin NURIANA, Nurfa ANISA and MARTANA
189
Measured and modeled plant growth and productivity in relation to SRI (System of
Rice Intensification) method on local paddy cultivar under salinity stress - Mu khammad
191
MURYONO and lndah TRISNAWATI
Diversity and speci es distribution of mangrove in East Java - Fari d K. MUZAKI, Dian
SAPTARIN I, Syaik hul MAHM UD, Muham mad ROM ADH ON I, Lind a Novit a SARI and Sofyan ARIS
193
Fun ctional biod iversity of Arthropods fa un a following seasonal variation in the
195
agri cultural landsca pe - lndah TRI SNAWATI and Mukhammad MURYONO
Study of Potential Phytoplankton as a Cause of HABs (Harmful Algal Blooms) in
197
Northern Coast of East Java- Dian SAPTARIN I, Fit ri Wu land ari EFFENDI, Wenny D. D.
RA HM ADIANI and Citra Fitri e RIANY
Toxi city test and ch e mical screening of Term inalia catappa leaf extract as a
199
bi ofungicide to control Phytopthora capsid growth on red pepper (Capsicum
frutescens longa .) - Dini ERMAVITALIN I, Kristan ti lndah PU RWANI , Tuti k NURHIDAYATI, Sri
URHATI KA
Optimisation of biosurfactant oroduction from Acinetobacter sp. P2(1) using glucose
201
substrate- FATIMAH, SUHARJONO, NI' MATUZAHROH, Tri ARDYATI, Afaf BAKTIR, Ahmad
HON TOW I
Characterize lypolitic and proteolytic bacteria from domestic sewage - Nita CITRASARI,
I' MATUZAHROH and Tri NURHARIYANTI
203
Isolation of HS Avia n Influenza viruses from domestic poultry in Surabaya, East Java,
205
In donesia, in 2010 - 2011 - Luh Ade Wilan KRISNA, Masaoki YAMAOKA, Aldise Mareta
ASTRI, Emmanuel Djoko POETRANTO, Laksmi WULANDARI , Resti YUDHAWATI, Landia
SETIAWATI, Retno Asih SETYONINGRUM, Teridah Ermala GINTING, Akiko MAKINO, Kyoko
_HI NYA and Yoshihiro KAWAOKA
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217
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219
Optimization of DNA extraction from seeds and fresh leaf tissues of Soybean (Glycine max)
Oeke YUNITA, Benny SETIAWAN and EVELYN
Faculty of Pharmacy
University of Surabaya
Ema il: oeke .ynita@gmail.com
Abstract
The effects of various components on extraction buffer such as SDS-NaCI, PVP, ｾ Ｍ ｭ･ｲ｣｡ｰｴｯｨ
ｮ ｯｬ
Ｌ＠
extraction with Phenoi:Chloroform :lsoamyl acetate, and incubation time on the DNA extraction from
seeds and fresh leaves of Soybean (Glycine max) were stud ied. Based on results above, an optimized
method for DNA extraction from Soybean seeds and leaves were established. Extracting Soybean
seeds twice with Phenoi :Chloroform:lsoamyl acetate (25:24:1), incubating for 30 min and 1% SDS-2
M NaCI in extraction solution could promote the quantity and purity of DNA from seeds, respect ive ly.
The results also showed th at high quality DNA from Soybean leaves could be extracted with
Phenoi:Chloroform:lsoamyl acetate (25:24:1) twice, incubating for 30 min and 0.5% SDS-2% PVP in
extraction solution, although the DNA quantity was less than DNA quantity obtained with
Nucleospin· Plant II method .
Keywords
:Glycine max, DNA extraction, optimization
INTRODUCTION
Soybean (Glycine max) is an important commodity in Indonesian Protective Commitment in
International Trading Confe rence (WT0)( 1 ) which is used in food and herbal supplement.(2)
Despite its important effect as herbal supplement, there were many reports that revealed its side
effects on skin, gastrointestinal, and respiratory reactions and in some cases anaphylaxisYl Before
further evaluation about its side effect, correct identification of the raw material must be performed
to ensure its safety. One of the most reliable methods for identification of herbal medicine materials
is by analyzing DNA. DNA markers are reliable for informative polymorphisms as the genetic
composition is unique for each species and is not affected by age, physiological conditions as well as
environmental factors. (4 )
The application of genetic identification in some plant species has however been constrained by lack
of efficient DNA extraction techniques, because of the presence of polyphenols and metabolites that
interfere with further application of DNA, such as DNA fingerprinting. (S) Therefore this study had
performed optimization of DNA extraction from seeds and fresh leaves of soybean .
MATERIAL AND METHODS
Plant material
Soybean seeds were collected from local market at Surabaya on 2011. After being cultivated several
days in the soil, the leaves from the seeds were collected, washed free of dirt, mopped dry and
quickly stored at -80 2C until used.
Solutions
The extraction buffer cons isted of 200 mM Tris-HCI pH 8.0, 250 mM NaCI, 25 mM EDTA, and 0.5 _
SDS was prepared. The following solutions were also prepared and stored
at
4•c
Phenoi:Chloroform: lsoamylacetate (25:24:1), 70% Ethanol.
DNA extraction
6
Genom ic DNA extraction, modified from ICI Seeds Co.1996( l. Seeds (Fresh leaves) 100.0 mg we re
ground and ＴＰｾＭｴｬ＠
of extraction buffer (200 mM Tris-HCI pH 8.0, 250 mM NaCI, 25 mM EDTA, anc
0.5% SDS) were added . The mixture was added w ith ＴＰｾＭｴｬ＠
phenol with occasi onal inversion and th er
centrifuged 12,000rpm 4•c for 10 min. Three hundreds micro littre of upper phase was mixed wi-ＲＵＰｾＭｴｬ＠
isopropanol and incubated at room temperature for 10 min. After centrifugation 12,000 g c: :
42C for 10m in, the DNA pellet was washed with
ＶＰｾＭｴｬ＠
4•c for 10 min and DNA pe llet was dissolved in ＳＰｾＭｴｬ＠
of 70% cold ethanol, centrifuged 12,000rpr
sterile water (for leaves) or ＱＰｾＭｴｬ＠
(for see s
and kept at -20 2( until used.
Optimization of DNA extraction from Soybean seeds and leaves
Based on extraction procedure above, following modifications on DNA extraction method
done
respectively:
(1)
with 2% PVP for leaves;
ｷ
･
ｾ＠
(2) DNA extraction with phenol, Phe nc
Ch loroform:lsoamyl acetate (25 :24:1) once and tw ice; (3) set a different incubation time (0 mi n,
3:
min, and 60 min); and (4) adjust the concentration of SDS in extraction solution to 0.5%, 1.0% a :
2.0%, respectively; (5) adjust the composition of SDS and NaCI (0.5% SDS- 250mM NaCI; 1% SDS-2'
NaCI) f o r seeds; (6) adjust th e concentration of 6-mercaptoethano/ (1.0%, 5.0%, and 10.0%) fc·
leaves.
Detection of quality and quantity of DNA from the seeds and leaves of Soybean
DNA
purity
(A260/A280
ratio)
and
its
concentration
were
measu red
with
Nano Drc:
spectrophotometer.
RESULTS and DISCUSSION
This study had used seeds and leaves of soybean fo r extracting the DNA. Soybean seeds were useo this study because seeds frequently being sold at the local market and they were usually cons ulT'