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C DNA

The document outlines the process of Reverse Transcriptase PCR (RT-PCR), which converts RNA to complementary DNA (cDNA) and amplifies it. It details the necessary reagents and a step-by-step protocol for synthesizing cDNA from RNA, including incubation times and temperatures. The final product can be used for further amplification through standard PCR techniques.

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8 views2 pages

C DNA

The document outlines the process of Reverse Transcriptase PCR (RT-PCR), which converts RNA to complementary DNA (cDNA) and amplifies it. It details the necessary reagents and a step-by-step protocol for synthesizing cDNA from RNA, including incubation times and temperatures. The final product can be used for further amplification through standard PCR techniques.

Uploaded by

uqd2022
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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cDNA preparation and RT-PCR

Reverse Transcriptase PCR (RT–PCR) is a one or two step process that is


undergone when converting RNA to DNA and the subsequent amplification of
the DNA that has been reversely transcribed. In the first step of RT-PCR, which
is called the first strand cDNA synthesis, the complementary DNA (cDNA) is
produced from an mRNA template that uses dNTPs and a reverse transcriptase.
The components mentioned are combined with a primer (Oligo (dT) or random
primer) in a reverse transcriptase buffer for approximately one hour at a
temperature of 37oC. When the reverse transcriptase reaction has been completed
a cDNA will have been produced from the original single stranded mRNA. At
this point standard PCR, or second strand reaction, is performed. In this two-step
RT–PCR, Taq DNA polymerase and the upstream and downstream DNA primers
are added. The reaction is further helped by putting it in a temperature that is
above 37oC will aid in binding DNA primers to the cDNA. The subsequent
higher temperatures will let the DNA polymerase to produce double stranded
DNA from the cDNA.

Reagents required
1. Total RNA (or mRNA)
2. Oligo (dT)18 primer
3. Reverse transcriptase
4. dNTPs
5. Taq DNA polymerase
6. RNase Inhibitor

Protocol
1. Transfer 1-3 μg of Total RNA (or 100 ng of mRNA) to a fresh microfuge tube.
DNase treatment is recommended to avoid DNA contamination.
Total RNA 1-3 μg
DNase enzyme 1 unit
DNase buffer 1x
260
Adjust the volume to 10μl with Nuclease free water. Incubate at 37oC for 30
mins. Add 25mM EDTA and incubate at 65oC for 10 mins. Chill on ice. Take 1-
3 μg of DNase treated RNA, Add following:
Total RNA 1-3 μg
Oligo (dT)18 primer 0.5 μg
Nuclease free water to make up the volume to 12.5 μl

Incubate at 65oC for 5 mins. This step denatures RNA and removes any secondary
structures.
2. To the denatured RNA add:
RT buffer 1x
dNTPs 20 mM
RNase Inhibitor 20 units
Reverse transcriptase 40 units
Nuclease free water to make up the volume to 20 μl

3. Incubate the reaction for 60 min at 42oC.

4. Inactivate the reverse transcriptase and denature the template-cDNA


complexes by heating the reaction to 70oC for 5 minutes.

5. The first strand cDNA can then be used to amplify a specific DNA fragment
by normal PCR reaction using specific sense and antisense primers.

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