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STAGE TEMPERATURE TIME

A.INITIALIZATION 95°C 3 min
B. AMPLIFICATION
1.DENATURATION 95°C 30 sec
2.ANNEALING 55°C 1 min
30 CYCLES
3.EXTENSION 72°C 2 min
C. FINAL 72°C 8 min
ELONGATION
AND HOLD 4°C ∞
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 Electrophoresis means “to carry with electricity”
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➢Serum proteins
➢Gel electrophoresis
SIZE
➢ GEL WEB

•DNA fragments are

negatively charged
•Opposites attract

Phosphate and
sugar backbone
In the gel polysaccharide (agarose) strands
form microscopic gaps
Proteins

DNA and RNA

 DNA has a net negative charge and will
migrate toward the anode (positive pole).

PCR products are loaded into an agarose

gel and electrophoresed.

MUTAGEN!!!!
Ethidium bromide is a dye that
intercalates into nucleic acids and will
fluoresce with an orange color under
ultraviolet (UV ) irradiation.
An image analyzer uses UV light to
capture computer images of the PCR
products.
1. Tank with power supply (electrical field)
2. Electrode, either carbon or platinum
3. Buffer system (in the buffer tanks)
4. Support media like :
- Agar gel
- Agarose
- Cellulose acetate
- Polyacrylamide
5. Stains - This is used to visualize the separate bands.
6. Densitometer to read the different bands and quantitate them.
WHAT DO YOU CAN WE USE THIS TECHNIQUE FOR?

Forensics
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For detecting protein For detecting protein For detecting DNA For detecting RNA
post-transnational modifications
(PTM)
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 PROBES

•Radioactive e.g. 32P •Non-radioactive e.g. Biotin

•Sensitive •Sensitive
•Relatively cheap •Relatively expensive
•Hazardous
Dr. Azhar Chishti

You should follow the

radioactive waste disposal
regulations.
Biotin Avidin *
Probe

Probe * Target DNA

Target DNA
 Hybridization

Dr. Azhar Chishti

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→
→
“IMMUNOBLOTTING/ PROTEIN BLOTTING”
➢ PROTEIN

→ protein
antibody
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APPLICATIONS OF WB:
❑ For HIV

❑ For HBV

❑ For Herpes

❑ Bovine spongiform encephalopathy (BSE)

❑ Lyme disease