ISOLATION OF DNA

Tianing Ni Wayan
DEPT. BIOCHEMISTRY
FACULTY OF MEDICINE
UDAYANA UNIVERSITY
 DNA IN THE CELLS

• Organelles contain the DNA:

 In the nucleus  nucleus/chromosomal
DNA (nDNA)
 In the cythoplasm Mitochondria DNA 
matrix (mtDNA)
 Chloroplast DNA
I. Chromosomal DNA
• Mammals  6 x 109
• Plants  2 x 108 - 2 x 1011
• Fungi  2 x 107 - 2 x 108

II. Mitochondrial DNA

• Animals 16 x 103 - 19 x 103
• Higher plants  150 x 103 - 250 x 104
• Fungi  17 x 103 - 78 x 103
• Green alga  16 x 103
• Protozoa  22 x 103 - 40 x 103
III. Chloroplast DNA
• higher plants  120 x 103 - 200 x 103
• green alga  180 x 103
TOOLS FOR ISOLATION OF
DNA
Rack-Tubes
Micropipettes
and Tips
BINDER
 EPPENDORF /
MICROCENTRIFUGE-TUBES

2 ml, 1,5 ml 200 ul

LAMINAR FLOW
Water-bath
CENTRIFUGES
• Glass baker
• Autoclaps
• Vortex
• Freezer (4OC, -20OC and -
80OC)
• Ect
Samples for isolation of DNA

 Whole Blood
 Hair
 Bone
 Another tissue
 Materials for isolation
1. BUFFER Red Blood Cell (RBC)
 Ammonium chloride (NH4HCl)
 Potassium bicarbonate (KHCO3)
 EDTA
 Aquadest
2. BUFFER Cell Lysis Solutions (CLS)
 Tris-HCl
 EDTA
 SDS
 Aquabidest
3. Ammonium acetat/Proteinase
4. RNase
5. Ethanol/Isopropanol
6. Alkohol
7. Tris-EDTA (TE)
Schema of DNA isolation
DETECTION OF DNA ISOLATION
• SPECTROFOTOMETRY
• OPTICAL DENCITY (OD) 260/280 nm
(1,90 – 2,0 µg/m/)
• ELECTROPHORESIS ON AGAROSA
GEL AND VISUALISATION WITH UV.
Tools for Electrophoresis
Gel Doc (BIO-RAD)
VISUALISATION
Polymerase Chain Reaction
(PCR)
 WHAT IS PCR
• To amplification or to make copies of
a gene for sequencing/analysis.
• There are done on an automated
cycler.
• Use the tubes for reaction mixture.
 STEPS OF PCR
I. Cycle I: Pre Denaturation: 94°C (1’) (1X)
II. Cycle II (30-35):
1. Denaturation at 94°C
To make the single strand of DNA.
2. Annealing at 50 - 54°C
3. Extension at 72°C
III. Cycle III: Final Extension at 72°C (5’) (1X)
 ELECTROPHORESIS
• TO NOW THE MOLECULAR WEIGH OF
THE GENE OR TO PURIFICATION OF
DNA.
• USE THE AGAROSA OR ACRILAMID
GEL.
 AGAROSA CONCENTRATION

• MW gel con.
• 1000 – 20.000 bp 0,6%
• 500 – 5000 bp 1%
• 100 – 2000 bp 2%
• 10 – 500 bp 3 – 4%
• POLIMER LINIER D-GALAKTOSA with L-
GALAKTOSA
• MORE EASY TO WORK THAN ACRILAMID
GEL
 BUFFER SYSTEM
• BUFFER:
Tris Acetat EDTA (TAE)
Tris Borate EDTA (TBE)
Tris Phosphate EDTA (TPE)
• TAE BUFFER: FOR THE LOW
MOLECULAR WEIGHT AND LOW
CAPACITY.
• TBE dan TPE: More expensive but
buffering capacity better than TAE.
• LOADING DYE
• ETHIDIUM BROMIDE
 RESTRICTION FRAGMEN LENGTH
POLYMORPHISM (RFLP)
• POLYMORPHISM: VARIATION OF GENETIC
MICRO SATELIT
Specific repeated sequence (Tandem
repeated)
Variable number of tandem repeated (VNTR)
Shot tandem repeat (STR)
Single nucleotide polymorphism (SNP)
• USED THE RESTRICTION ENDONUCLEASE
ENZYME
To cut the specific sequence of DNA.
• THANK
YOU