2. 1. Reaction of DNA replication in vitro.
2. To identify DNA genom by means of amplifying
DNA segment (part of the genom) using
specific primer compatible to DNA of interest.
3. To synthesize DNA mediated by primer extention
in repeat cycles.
3. Basic Principle :
- Apply pair of primers complementary to 3’ end of DNA
segment & allow DNA polymerase to extend the
primers through the addition of dNTPs DNA
synthesis proceeds across the region between the two
primers.
- Repeat the reaction several times (cycles) to obtain
sufficient amplified DNA.
4.
5. Methods
- Prepare reaction mixture in PCR tube :
10 x reaction buffer 5 ul
dNTPs (12.5 mM) 1 ul
upstream primer 10 pmol/ml 1 ul
downstream primer 10 pmol/ml 1 ul
Taq DNA polymerase 0.5 ul
sterile destilled water 39.5 ul
______
total reaction mixture 50 ul
6.
7. - Design program for specific amplification in thermal
cycler (PCR machine).
Ex. PCR program for FSH receptor gene (35 cycles)
1st denaturation 94 C for 4 minutes
cycle denaturation 94 C for 45 seconds
primer annealing 55 C for 45 seconds
primer extension 75 C for 1.5 minutes
the last cycle is extended 72 C for 5 minutes
- Load PCR mixture on thermal cycler and incubate in
design program condition as above.
- Remove PCR mixture & analyzed the results by gel
electrophoresis.
11. Basic principles
- Separation of molecule mixtures based on the different
of their charge, melecular weight (size) & shape.
- DNA is negatively charge move from cathode to
anode pole in electric field.
- The movement is proportional to their molecular weight
12. Equipments : electrophoretic chamber
electrophoretic tray
power supply
Method
- Prepare 0.8% agarose in 1 x TAE (Tris acetic
acid EDTA) buffer melt it by boilling to 100 C for a
minute.
- Poor the gel to electrophoretic tray that have been
prepared with comb to create wells on one side end
of the gel.
- Allow the gel to solidify in room temperature.
14. - Put the gel in electrophoretic chamber filled with 1 x
TAE buffer the gel is soaked (covered) in buffer.
- Mix DNA samples with tracking dye (bromophenol blue
+ xylene xianol) & load to the well by pippetting gently.
- Connect the chamber to power supply & set the
voltage at 90 V for an hour.
- Remove the gel & stained with ethidium bromide
DNA seperation is examined under UV transilluminator
- Take the picture using polaroid camera.
