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PCR Shortly Explained
PCR Shortly Explained
PCR Shortly Explained
Taq polymerase
How can you get pcr product with use of regular dna polymerase?
Dntp (nucleotides)
Each dNTPs should be present in equimolar concentration.
The concentration of dNTPs in PCR reaction should be around 50200M.
If the concentration of dNTPs is high the fidelity of the process will be
adversely affected by driving Taq DNA polymerase to misincorporate
at a higher rate than normal
While if the concentration is lower it may affect efficiency of PCR.
Buffer solution for providing optimum activity and stability of dna
polymerase.
The general composition of 10X buffer: -100 mM Tris H-VI (pH 8.3 at
250C ) 500 mM KCl+15mM MgCl2 +1mg/ml gelatin+0.1% tween
20+0.1% NP40
Divalent cations are used for mutagenesis (eg. Mg2+ , Mn2+)
Exists as dNTP-Mg2+ complexes,
Interacts with DNA backbone ( what it actually does by interacting
with backbone?)
Essential cofactor for Taq polymerase
The total magnesium ion concentration must exceed the total dNTP
concentration, for it to act as cofactor.
Low concentrations tend to have low yields of PCR product.
High concentrations tend to reduce the fidelity of Taq polymerase and
lead to amplification of non-specific products.
Monovalent cation- potassium ions.
Other Components
The salt used in most reactions is K / Na, added to facilitate correct
primer annealing.
Other components stabilize the enzyme are: gelatin, bovine serum
albumin or nonionic detergent(Tween 20).
Lecture 12-13
Cloning methods