Sei sulla pagina 1di 12

DIAGRAM ALIR

EKSTRAKSI DNA DENGAN BUFFER ALKALI

1. (Yahya et al., 2017) Comparison of DNA Extraction Methods Between Conventional, Kit, Alkali
and Buffer-Only for PCR Amplification on Raw and Boiled Bovine and Porcine Meat
Modified Buntjer et al, 1995

80g & 150g sample

2 ml NaOH 0,5 M per g

Vortex

Incubated at 100OC, 10 minutes

Centrifuged at 13.000 rpm for 2 minutes at 37°C twice


Natan

Supernatan Transfer into new microtube

5 M ammonium acetate
at 0.5 times volume

Absolute ethanol 2-3 times

Incubated at -20OC, 15 minutes

Centrifuged for at 13.000 rpm, 5 minutes

Pellet

500 μL of 70% ethanol

Centrifuged for at 13.000 rpm, 5 minutes

100 μL TE buffer

Supernatan

Materials
2 ml NaOH (0,5 per gram)
5 M ammonium acetate needs 0,5 times volume
Absolute ethanol needs 2-3 times volume
500 μL of 70% ethanol
100 μL TE buffer

Equipment
1,5 mL microtube
Micropipette
Vortex
Incubator thermomixer
Centrifuge
2. (Yalçınkaya, 2016) Comparison of DNA extraction methods for meat analysis
Modified Lenstra et al, 2001
50 mg homogenate sample

600 μl of lysis buffer:


0,5 M NaOH, 10 mM EDTA

Incubated at -80OC, 5 minutes

Vortex

Incubated at 100OC, 7 minutes

Centrifuged at 12.000xg, 2 minutes


Natan

Supernatan Transfer into new microtube

200 μL of 2 M ammonium acetate

Vortex 20 s

Incubated for 5 min on ice

Centrifuged at 14.000xg, 3 min, 4OC


Natan

Supernatan Transfer into new microtube

600 μL of isopropanol

Gently mixed up and down 10 times

Centrifuged 14.000xg, 2 min


Supernatan

Natan

600 μL of 70% ethanol


Materials
0,5 M NaOH Centrifuged 14.000xg, 2 min
10 mM EDTA
200 μL of 2 M ammonium acetate Drying
600 μL of isopropanol 100 μL TE buffer
600 μL of 70% ethanol
100 μL TE buffer Supernatan

Equipment
Microtube
Micropipette
Vortex
Incubator thermomixer
Centrifuge
3. (Lenstra et al 2001) On the origin of meat - DNA techniques for species identification in meat
products
a. Rapid alkaline extraction of DNA from meat
Materials
∙ Extraction buffer: 0.5 M NaOH, 10 mM EDTA
Method
1. Weigh 100 mg tissue in a 2-ml Eppendorf tube.
2. Add 0.2 ml extraction buffer, vortex and incubate at 100°C for 7 min.
3. Centrifuge for 2 min at 12,000 rpm and transfer supernatant to a new tube, avoiding floating fat.
4. Repeat step 3.

b. Alkaline and organic extraction of DNA from meat


Materials
∙ Extraction buffer: 0.5 M NaOH, 10 mM EDTA
∙ Alkaline solution: 2 M NaOH
∙ Phenol chloroform isoamylalchohol (PCI) solution:
0.05 % (w/v) hydroxyquinoline,
2 % (v/v) isoamylalcohol,
49 % (v/v) phenol,
water-saturated
neutralised with Tris/HCl [13],
49 % (v/v) CHCl3
Method
1. Weigh 1 g ground tissue into a 10-ml tube.
2. Preheat extraction buffer to 100°C, add 2 ml to the tissue and incubate at 100°C for 7 min.
3. Centrifuge at 10,000-15,000 rpm for 2 min and transfer the supernatant to a fresh tube, avoiding the
floating fat layer.
4. Centrifuge at 10,000-15,000 rpm for 2 min and transfer 100 ml supernatant to an Eppendorf vial.
5. Add 50 ml PCI solution and leave for 5 min, vortexing occasionally.
6. Centrifuge at 10,000-15,000 rpm for 3 min in a tabletop minifuge and transfer the (upper) aqueous
phase to a new Eppendorf vial.
7. Add 25 µl 2M NaOH to the aqueous phase.

4. (Buntjer et al, 1995) Rapidspecies identificationin meat by usingsatelliteDNA probes


- DNA alkaline extraction from meat.
DNA was extracted by heating 100-400 mg of the meat samples at 100OC in 2ml of 0.5 M NaOH per
gram of meat for 7 min.
This extract was centrifuged at 13000g for 2 min and the supernatant was transferred to a new tube.
After a second centrifugation under the same conditions, the supernatant was spotted onto hybridization
membranes.
- Additional purification of alkaline DNA extract.
In somecases, alkaline DNA extractions were extracted once with 0.5 sample volume of a mixture of
phenol, chloroform and isoamyl alcohol (25:24:1, by vol) as described by Sambrook et al.
(Andika Ardi, 2012) VALIDASI METODE EKSTRAKSI DNA PADA ANALISIS DNA BABI DALAM
PRODUK BAKSO

Proses Pembuatan Bakso


Daging sapi segar yang sudah dibersihkan lemaknya dipotong kecil-kecil,
kemudian ditambahkan daging babi (0.1, 0.5, 1, 5, 10, 15, dan 20% b/b) dan
dimasukkan ke dalam food processor.
Setelah itu, dimasukkan bahan tambahan seperti es batu, tepung tapioka, garam, bawang putih, natrium
tripolifosfat (STPP), dan merica dengan komposisi masing-masing 40, 20, 15, 10, 5, dan 0.5% dari total
bobot daging.
Adonan selanjutnya dicetak berbentuk bulatan-bulatan bakso (seberapa) dan
dimasukkan ke dalam panci yang berisi air mendidih (80oC) selama 15 menit.
Bulatan-bulatan bakso dimasak kembali pada air mendidih (100oC),
lalu diangkat dan ditiriskan selama 15 menit.
Bakso dikemas dalam plastik untuk selanjutnya diekstraksi.

Ekstraksi DNA
Ekstraksi DNA pada sampel dilakukan dengan menggunakan metode standar fenolkloroform
(Sambrook et al. 1989) yang telah dimodifikasi untuk produk tercemar daging babi.
Preparasi Sampel
Sebanyak 75 mg sampel bakso diambil dari 5 titik yang tersebar pada sampel.
Selanjutnya sampel dimasukkan ke dalam tabung eppendorf 1.5 mL.
Degradasi Protein
Sampel dalam tabung ditambahkan 1×STE sampai volume 340 µL, 40 µL SDS 10%, dan 20 µL protein
kinase K 5 mg/mL.
Campuran diinkubasi pada suhu 55oC selama 2 jam sambil digoyang perlahan.
Degradasi Bahan Organik
Sampel yang telah diinkubasi ditambahkan 400 µL larutan fenol, 400 µL kloroform-isoamil alkohol (24:1),
dan 40 µL NaCl 5 M.
Campuran digoyang dalam suhu kamar selama 1 jam.
Presipitasi DNA
Sampel hasil ekstraksi selanjutnya disentrifugasi pada kecepatan 12 000 rpm selama 5 menit hingga fase
air terpisah dengan fase fenol.
Fase air dipindahkan ke dalam tabung baru dengan volume terukur (± 400 µL).
Molekul DNA diendapkan dengan cara menambahkan 800 µL alkohol absolut dan 40 µL NaCl 5 M.
Campuran kemudian diinkubasi pada suhu -20 oC selama semalam, dan selanjutnya disentrifugasi pada
kecepatan 12 000 rpm selama 1 menit.
Endapan DNA yang diperoleh dicuci dengan alkohol 70% kemudian diendapkan lagi.
Endapan DNA yang telah bersih dari alkohol dipulihkan dengan menambahkan 100 µL TE. Sampel DNA
disimpan pada suhu -20 oC dan siap digunakan.
Pengujian DNA Total
Pengujian DNA hasil ekstraksi secara kualitatif dilakukan dengan elektroforesis pada gel agarosa 1%.
Gel dibuat dari 0.3 g agarosa dan 30 mL larutan bufer (0.5 × TBE) yang dipanaskan. Larutan agarosa
dibiarkan agak dingin sambil diaduk dengan pengaduk magnet, lalu ditambahkan 1.25 μL pewarna etidium
bromida. Sebanyak 5 μL sampel DNA dilarutkan dalam 1 μL loading dye, lalu elektroforesis dilakukan
selama 40 menit pada tegangan konstan 100 volt sampai pewarna mencapai bagian bawah gel. Pengujian
DNA hasil ekstraksi secara kuantitatif dilakukan dengan spektrofotometer GeneQuant 1300. Sampel DNA
3 μL dimasukkan ke dalam tabung eppendorf 1.5 mL dan ditambahkan akuades 597 μL. Larutan TE
digunakan sebagai blangko, dibuat dengan cara yang sama, yaitu sebanyak 3 μL larutan TE ditambah
597 µL akuades, lalu dimasukkan ke dalam tabung eppendorf 1.5 mL. Sampel dan blangko di-spin down
selama 0.5 menit, kemudian dilakukan pengujian dengan spektrofotometer
Rapid alkaline extraction of DNA from meat (Lenstra, 2001)

100 mg tissue

Weigh in a 2-ml Eppendorf tube


0,2 ml extraction buffer:
0,5 M NaOH, 10 mM EDTA

Vortex

Incubated at 100OC, 7 minutes

Centrifuged at 12.000xg, 2 minutes


Natan

Supernatan Transfer into new microtube

Centrifuged at 12.000xg, 2 minutes


Natan

Supernatan Transfer into new microtube

Centrifuged at 12.000xg, 2 minutes


Natan

Supernatan Transfer into new microtube

Materials
0,5 M NaOH
10 mM EDTA

Equipment
Microtube
Micropipette
Vortex
Incubator thermomixer
Centrifuge
Alkalin lysis for buffalo
P. S. Girish & S. Haunshi & S. Vaithiyanathan & R. Rajitha & C. Ramakrishna (2013)

500 mg of meat sample

four volumes of 0.2 N NaOH

triturated in pestle & mortar

transfered in a sterile micro centrifuge tubes

four, six, eight & ten volumes


of 0.2N NaOH i.e. in 20, 30,
40 & 50 μl of 0.2N NaOH

heated at 75 °C in water bath for 20 min a

180, 270, 360 & 450 μl of 0.04


M Tris HCl (pH 7.75)

DNA extract
Protocol B: DNA extraction using NaOH
E.M. Santos1 , J.F.R. Paula1 , P.M.C. Motta1 , M.B. Heinemann1 , R.C. Leite1 , J.P.A. Haddad1 , H.L.
Del Puerto2 and J.K.P. Reis1 (2010)

100 µL PBMC and macerated lung fragment

400 µL TNE:
0,1 M Tris, pH 8.0
0,15 M NaCl
0,05 M EDTA
Washed

Centrifuged at 10,500 g, 3 minutes


Natan

Supernatan Transfer into new microtube

40 µL lysis solution
(50 mM NaOH)

resuspended

Vortex 5 minutes

Heated in waterbath at 95OC, 5 minutes

15 µL 0,50 M Tris-HCl, pH 8

Resuspended, 2 minutes

Stored at -20OC

Supernatan
BUFFERS AND REAGENTS FOR DNA EXTRACTION
Phosphate Buffered Saline (PBS) pH 8.0
Di-sodium hydrogen phosphate 13.48 g
Sodium di-hydrogen phosphate, hydrated 00.78 g
Sodium Chloride 04.05 g
Distilled water to make 1000 m l

Lysis buffer (fresh meat) pH 8.0


Tris hydrochloride, 10 mM 121 mg
EDTA, 0.1 M 3.722 g
SDS, 0.5 % 0.5 g
RNase (DNase free, pancreatic) 20 μg/ml
Distil water to make 100 ml

Lysis buffer (cooked meat) pH 8.0


Tris-HCl, 10 mM 121 mg
NaCl, 100 mM 0.584 g
EDTA, 5 mM 0.1861 g
SDS , 1 % 1.0 g
Di-thiothretitol (DTT), 10 mM 0.15424 g
Distil water to make 100 ml

DNA extraction buffer (for blood)


Tris buffer (pH 8.0), 1M 5 ml
Sodium chloride, 5 M 40 ml
EDTA buffer, 0.5 M 2 ml
Distilled water up to 500 ml
(Autoclave in batches of 100 ml)

Sodium dodecyl Sulphate (SDS) 10%


SDS 1g
Distilled water to make 10 ml

Chloroform: Isoamyl alcohol preparation (24:1)


Chloroform 24 ml
Isoamyl alcohol 1 ml
Mix thoroughly and store at 4oC

Phenol: Chloroform: Isoamyl alcohol preparation (25: 24:1)


Tris saturated phenol 25 ml
Chloroform- Isoamyl alcohol (24:1) 25 ml
Mix thoroughly and store at 4Oc

70 % alcohol
Absolute alcohol 30 ml
Distilled water up to 100 ml and mix thoroughly

Ammonium acetate (10 mM)


Ammonium acetate 77.08 mg
Distilled water (up to) 100 ml
Tris-EDTA (TE) buffer 1X, pH 8.0
Tris HCl, 10 mM 121.14 mg
EDTA, 1 mM 37.22 mg
Distilled water up to 100 ml and adjust pH 8.0

Ethylene diamine tetracetate (EDTA), 0.5 M, pH 8.0


EDTA 18.612 g
Distilled water 80 ml
Stir vigorously on a magnetic stirrer to mix.
Adjust the pH to 8.0 and make the volume to 100 ml.
Autoclave and store

Sodium hydroxide (NaOH) 10 N


Sodium hydroxide 4g
Add distilled water to make 10 ml

Tris-HCl 1 M,. pH 8.0


Tris-base 121.1 g
Dissolve in distilled water 800 ml
Adjust the pH to 8 by adding conc. HCl.
Make the volume to 1000 ml. Autoclave and store

Sodium chloride 5 M
Sodium chloride 29.22 g
Distilled water up to 100 ml
Sterilized by autoclaving

BUFFERS AND REAGENTS FOR AGAROSE GEL ELECTROPHOROSIS


TAE buffer (50X)
Tris base 12.1 g
Glacial Acetic acid 2.8 ml
0.5 M EDTA (pH 8.0) 5 ml
Distilled water (up to) 50 ml

TAE buffer (1X)


TAE buffer, 50 X 2 ml
Distilled water up to 100 ml

Ethidium bromide (10 mg/ml)


Ethidium bromide 10 mg
Distilled water 1 ml

Gel loading dye (6x)


Bromophenol blue 2.5 mg
Sucrose 500 mg
Distilled water 1 ml
Store at 4oC
Metode urea
50 mg homogenate sample

500 µl of the lysis buffer (10 mM TrisHCl, 20 mM


EDTA, 2% (m/v) SDS, 6 M urea, pH 8.0)

0
Incubated for 5 min at -80 C

vortex
2 µl of 20 mg/ml
Proteinase K
0
Gently shaken 15 min, 55 C, 1,200 rpm

50 µl of 5 M NaCl

500 µl of P:C:I
alcohol (25:24:1)
vortex

Centrifuged for 5 min at 10,000 rpm

supernatant transferred into a new tube


330 µl of isopropanol

Centrifuged for 5 min at 10,000 rpm


100 µl of TE buffer

pallet
Salt Method
50 mg homogenate sample

400 µl of the lysis buffer (10 mM Tris-HCl, pH 8.0,


2 mM EDTA, pH 8.0, 0.4 M NaCl) and in 40 µl of
20% (w/v) SDS

Vortex
2 µl of 20 mg/ml
Proteinase K

0
Incubated for 1 hour at 65 C

300 µl of 6 M NaCl

Vortex 30 sec

Centrifuged for 30 min at 10,000 rpm


pellet

Supernatant
330 µl of isopropanol

0
Incubated at 20 C for 10 min

Centrifugated for 20 min at 16,000 rpm


pellet 100 µl of TE buffer

pellet