Restriction Fragment Length Polymorphism

(RFLP) Analysis of Staphylococcus aureus using

EcoR1 and HaeIII Enzymes

Group 3
Ari Ardiantoro
(135090101111004)
Alifia Issabella Mulyawati
(135090107111001)
Esti Rizkiana Pratiwi
(135090101111044)
Zahrina Zata Dini
(135090101111011)
Department of Biology
Faculty of Mathematics and Natural Sciences
Brawijaya University

MATERIAL AND METHODS

INTRODUCTION

Identification method ?
Organism diversity
RFLP
(Restriction Fragment
Length Polymorphism)

MATERIAL
AND
METHODS
INTRODUCTION
Problems
1. How to analyze the band profile after digest by
restriction enzymes EcoRI and HaeIII ?
2. How to detect polymorphism in 16S rDNA gene by
RFLP method?
Objectives
1. Analyze the band profile after digest by restriction
enzymes EcoRI and HaeIII
2. Detection of polymorphism in 16S rDNA gene by
RFLP method
Benefits
Species identification based on DNA sequences to know
the diversity of organisms. It also useful to find where a
specific gene for a disease lies on a chromosome.

MATERIAL AND METHODS

MATERIALS
AND METHODS
Preparation for 10 L total reaction :
dH2O 4 L, digest buffer 1 L,
DNA 4 L, restriction enzyme 1 L
Spindown

Incubation at 70 C 15
min

Incubation at 37 C 1
hour

MATERIAL
AND
METHODS
ELECTROPHORESIS

Loading dye was added to

restriction digestion fragments

The gel was analyzed in

the UV transilluminator
and polaroid camera

Loaded on 1.5 % agarose

gel

Running for 45 min; 50 V

RESULT

Figure 2. The RFLP analysis of Staphylococcus aureus 16S

rDNA gene. The PCR amplified fragments of DNA cleaved by:
EcoR1 PCR-RFLP (1E), EcoR1 RFLP (2E, 3E and 4E) and
HaeIII PCR-RFLP (1H), HaeIII RFLP (2H, 3H and 4H). K1
as negative control for EcoR1 and K2 as negative control for
HaeIII.

MATERIAL
AND
METHODS
DISCUSSION

b
(Konnai et al., 2003)
Figure 2. The PCR-RFLP analysis of exon 2 of the Ovar-DRB1
gene (296 bp) from seven Suffolk sheep, B.0248, W.199, W194,
B.0240, IW.279, or.104 and W.196. The PCR-amplified fragments
of DNA cleaved by: (a) HaeIII and (b) EcoR1

MATERIAL
AND
METHODS
DISCUSSION

HaeIII

EcoR1

MATERIAL
AND
METHODS
TROUBLESHOOTING
Table 1. Technical practices troubleshooting (Jagielski
et al., 2014)
No.
1.

2.

Problems
Partial or no
digestion

Possible Cause

Solution

Insufficient incubation
time

Incubate the samples for longer time

at 37 OC (60-120 minutes)

Improper addition of
restriction enzyme

Always add the restriction enzyme at

the end of the reaction mixture and
add appropriate amount

Degradation of
restriction enzyme

Always place the vials containing

restriction enzymes on ice

Improper
Gel not run for
resolution of
sufficient duration
electrophoretic
separation of
fragments

Run the gel for longer period of

time till the bands are separated
properly

CLOSING
Conclusion
1. There was no bands appeared after electrophoresis,
because the restriction enzymes fail to digest
samples sequences
2. The polymorphism can not be analyzed or there is no
polymorphism because the bands were not appeared.
Suggestions
1. Incubate the samples + restriction enzyme for longer
time at 37 OC (60-120 minutes)
2. Run the gel electrophoresis for longer period of time
till the bands are separated properly

MATERIAL
AND
METHODS
REFERENCES
Jagielski, T., J. V. Ingen, N. Rastogi, J. Dziadek, P K. Mazur and
Jacek Bielecki. 2014. Current methods in the molecular
typing of Mycobacterium tuberculosis and other
Mycobacteria. Hindawi Publishing Corporation BioMed
Research
International. 1-21.
Konnai, S., Y. Nagaoka, S. Takesima, M. Onuma and Y. Aida.
2003. Technical note: DNA typing for Ovine MHC DRB1
using polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP). Journal Dairy Science. 83:33623365.

THANK YOU FOR YOUR ATTENTION

QUESTIONS ??
?