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8.
RT – PCR
Reverse Transcriptase PCR (RT-PCR) is a method used to amplify cDNA copies
of RNA. RT-PCR is used to retrieve and clone the 5’
and 3’ termini of mRNAs and to generate large cDNA
libraries from very small amounts of mRNA. This method can be used to know
the expression of the cloned genes. In addition, RT-PCR can be easily
adapted to identify mutations and polymorphisms in transcribed sequences and
to measure the strength of gene expression when the amounts of available
mRNA are limited and/or when the RNA interest is expressed at very low
levels. In RT-PCR both cDNA synthesis and PCR are performed in a single
tube using gene specific primers and target RNAs from either total RNA or
mRNA.
Components of RT PCR
1.
mRNA
2.
Oligo dT
3.
dNTP mix
4.
Buffer
5.
Reverse
transcriptase
6.
RNase
inhibitor
7.
Taq DNA
polymerase
8.
A set of
primers
All the
above components are taken in appropriate concentrations in a
microcentrifuge tube and kept in a thermal cycler which is programmed as
follows:
Step 1
cDNA synthesis – 45-550 C for 30 min.
Step 2
Denaturation 950 – 30 sec.
Step 3
Annealing 30 sec at 100 C below melting temp. of the
primers used
Step 4
Extension 720 C for 1-30 min.
Step 5
Goto 2 34 times
Step 6
40
Step 7
End
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