REPLIKASI

PCR
DENATURASI
(SUHU 90-95c)

ANNEALING
(SUHU 50-55c)
DNA PRIMER
POLYMERIZATION
(SUHU 70-72c)
Taq polymerase
none

none
Handout Bioteknologi Farmasi Dr. Oeke Yunita

Visualizing the DNA of Wolbachia sp.

DNA ladder

1 2 3 4 5 6 7 8

DNA ladder

wells

5,000 bp
2,000
1,650
1,000
850
650
500
400
300
200
100

1.
2.

Sampel mana yang positif untuk DNA Wolbachia?

Berapa bp ukuran fragmen DNA yang menjadi penanda Wolbachia sp.?

SAMPEL
-Bagian tanaman
-Cairan Biologis
-Sel/Jaringan

Isolasi DNA
Analisis Kualitatif (Kemurnian DNA)
- Spektrofotometri
(A260/A280) = 1,8-2,0
- Elektroforesis
cut by restriction enzmye

Analisis Kuantitatif (Spektrofotometri)

(A260 x faktor pengenceran x 50) ng/l

Amplifikasi DNA
(PCR method)

Elektroforesis
Hasil PCR

ANALISIS
DATA

What is Gel Electrophoresis?

Electro- = flow of electricity,
-phoresis = to carry across
A gel is a colloid, a suspension of tiny particles in a
medium, occurring in a solid form (like gelatin)
Gel electrophoresis refers to the separation of
charged particles located in a gel when an electric
current is applied
Charged particles can include DNA, amino acids,
polypeptides, etc

Gel Electrophoresis
Gels can be made from substances such as
agarose or polyacrylamide.
Agarose a complex sugar chain from red
seaweed. It is commonly used in foods (ice
cream, whipped cream, and jellies) and many
biological mediums. It has a large pore size
good for separating large molecules quickly.

Polyacrylamide chain of acrylic acid

Red Sea
Weed

molecules. It is often used to make plastics and

rubber. It has a small pore size good for
separating small molecules slowly.
*Polyacrylamide is a neurotoxin!
Acrylic Acid

DNA Ladder Standard

12,000 bp
5,000

DNA
migration
Note: bromophenol
blue migrates at
approximately the
same rate as a 300
bp DNA molecule

bromophenol blue
+

2,000
1,650
1,000
850
650
500
400
300
200
100

Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to
determine the sizes of unknown DNAs.

Fingerprinting Standard Curve: Semi-log

Molecular Weight
Determination
Distance (mm)

23,000

11.0

9,400

13.0

6,500

15.0

4,400

18.0

2,300

23.0

10,000

Size, base pairs

Size (bp)

100,000

1,000

100

2,000

24.0

10

15

Distance, mm

20

25

30

Differences between people

Person 1

cut sites

cut sites

GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
Person 2: more repeats

GCTTGTAACGGCCTCATCATCATCATCATCATCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA

1
DNA fingerprint

DNA
person 1

person 2

PCR APPLICATIONS
Comparing DNA samples from different
organisms to measure evolutionary
relationships
Comparing normal allele to disease allele
(Medical diagnostic)
Comparing DNA sample from crime scene
with suspects & victim (Forensics)
Paternity assay
Identifying Identity of Plant / microbe
species/variety QC method