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Introduction
Off all techniques In molecular biology, the polymerase
chain reaction (PCR), has been by for the most useful.
A basic PCR components and reagents A basic PCR components and reagents
DNA template: is the DNA molecules that contains the DNA
region (segment) to be amplified, the segment that we are 1) DNA template
concered with is the target sequence.
Two primers: a short segment of DNA that are complementary to 2) Two primers
the 3' ends of each of the sense and anti-sense strand of the DNA
target, they are needed to get DNA synthesis started. 3) Taq polymerase
4) Deoxynucleoside
triphosphates
Taq polymerase: is the enzyme to manufacture the DNA copies.
The PCR involves a couple of high temperature steps so we use a 1) Buffer solution
heat resistant DNA polymerase, this is extracted from heat
resistant bacteria living in a hot springs at temperature up to 80C, 2) Divalent cations
or another DNA polymerase with a temperature optimum at
around 70 C. 3) Monovalent cation
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PCR Procedure
PCR Procedure (Denaturation) The DNA to be amplified is mixes with
The mixture is heated to break the hydrogen bonds in Deoxynucleoside triphosphates, a thermal stable DNA
the DNA, forming single stranded molecules. polymerase and DNA primers.
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General Precautions:
There should be a spatial separation of PCR set up areas PCR Optimization:
from areas of purification and analysis of PCR.
The primer designing is very important in improving
the product yield and avoiding formation of spurious
products.
Use of pipette tips with filters (to minimize
contamination).
The choice of buffer or polymerase enzyme can help in
the amplification of long or otherwise problematic
storage of materials used in PCR should be separated regions of the DNA.
from all other reagents and should be added to the
reaction mixes in a sterile spatially separated facility.
General Precautions:
Use of PCR grade distilled water which has been heat and
UV sterilized .
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