pH is:
The negative value of Log [H+].
5 Buffers in body
(1) bicarbonate buffer (HCO3–/CO2),
(2) hemoglobin buffer (in erythrocytes),
(3) phosphate buffer,
(4) Acid protein buffer
Normal values of bicarbonate, PH, PCO2, PO2
HCO3: 22-26
PH: 7.35-7.45
PCO2: 35-45
PO2: 80-100%
4- Low ionic strength buffers give sharp bands and cause heat production so may denature protein
True or false?
false
What are the most common supporting media in electrophoresis
Agarose gel and Polyacrylamide gel
Enumerate five stains used in electrophoresis
- Bromophenol blue for proteins .
- Ninhydrin for amino acids .
- Sudan black for lipoproteins .
- Nitroblue tetrazolium for isoenzymes .
- lodine for polysaccharides
Factors affecting the velocity of migration of molecules
Electric field
Net charge of molecule
Friction coefficient
Buffers
Temperature
Three vectors are
Plasmid: double stranded circular; for small DNA fragments (10kb)
Phage: virus, for large DNA fragments (20kb)
cosmid: synthetic vector: large DNA fragments (50kb)
Requirements of PCR
a- Two oligonucleotide primers .
b- Thermus aquaticus DNA polymerase ( Taq polymerase ) .
c- All the four dNTP’s
d- DNA to be amplified.
e- Buffers.
Application of PCR
1- It allows synthesis of DNA for sequencing or cloning .
2- Diagnosis study and of genetic disease e.g. sickle cell anemia .
3- Diagnosis of viral disease as HIV .
4- Synthesis of human proteins ( e.g. insulin ) , synthesis of vaccines ( e.g.
hepatitis B virus) and synthesis of antibodies ( e.g. monoclonal antibodies ) .
5- Gene therapy .
6- Forensic analysis .
enumerate 5 supporting medium
Paper
Cellulose acetate
Agar
Agarose gel (common)
Polyacrylamide gel (common)
PCR temp for: Denaturation, Primer annealing. elongation
95C, 50C, 72C
The greatest buffering capacity at physiological pH would be provided by a protein
rich in which of the following amino acids?
Histidine
A climber attempts an assault on a high mountain in the Andes and reaches an
altitude of 5000 meters (16,400 ft) above sea level. What will happen to his arterial
PCO2 and pH?
The pH will rise and PCO2 will fall
DNA cloning is used for
Amplification of DNA in vivo
Process of cloning
- Plasmid is isolated then cut with a restriction enzyme forming cohesive ends .
- The foreign DNA is cut by the same restriction enzyme .
- The cohesive ends of the foreign DNA anneal with the ends of the plasmid DNA and joined by DNA ligase to form a recombinant plasmid DNA .
- The recombinant plasmid DNA is introduced into bacteria by transformation .
- The bacterial cells are allowed to divide and the recombinant plasmid DNA undergoes replication and amplification.
Metabolic acidosis is compensated by the kidney, true or false?
false
Separation of molecules in electrophoresis is according to their charges and
molecular weight. true or false?
True
Importance of buffers in electrophoresis
transmit electric current, adjust the pH and facilitate
migration of the substance.
enumerate 3 mechanisms for regulation of pH
Buffers - respiration - kidney
2 Methods for composition of buffers
A weak acid and its salt with strong base - A weak base and its salt with strong acid
Advantages of bicarbonate buffer system
- Present in higher concentrations than other buffers
- Easily formed at the tissues
- Easily corrected by respiration;
1 physiological buffer linked to the kidneys & another 1 linked to the lungs
Phosphate - Bicarbonate buffer system
The components of the electrophoresis unit.
- Electrodes: Cathode (-ve) and anode (+ve) made of platinum better than stainless
- Buffer reservoir
- Support of electrophoretic medium
- Transparent cover to prevent evaporation of the buffer
