cDNA SYNTHESIS
Content
Introduction .............................................................................................................................................2
Materials for cDNA Synthesis ................................................................................................................2
Method for cDNA Synthesis ....................................................................................................................3
Amplification of Single Strand cDNA by PCR ........................................................................................4
Mix the following components.................................................................................................................4
Perform PCR in Thermal Cycle at following conditions .........................................................................5
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cDNA SYNTHESIS
Introduction
Complementary DNA (cDNA) is a copy of DNA synthesized from mRNAs. During the reaction
Reverse Transcriptase Enzyme is utilized. Reverse Transcriptase is an RNA-dependent DNA
polymerase. The Enzyme cannot start DNA synthesis without RNA template which binds to single
strand mRNA sequence. cDNA synthesis experiment should be performed on ice.
Materials for cDNA Synthesis
 Template RNA
 Primer mix
 RNase free water
 Reaction buffer
 RNase inhibitor
 dNTP mix
 Reverse transcriptase
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cDNA SYNTHESIS
Method for cDNA Synthesis
 1 μL of oligodT primer is added to Template RNA (0.1 ng to 5 μg)
 RNase free water is added up to 12 μL of final volume
 Vortex the components and spin down
 Incubate the mix at 65°C for 5 minutes
 Place the tube on ice immediately after incubation
 Add 4 μL of Reaction Buffer
 Add 1 μL of RNase inhibitor
 Add 2 μL of dNTPs
 Add 1 μL of Reverse Transcriptase enzyme
 Total volume should be 20 μL.
 Pipet the mix carefully and centrifuge briefly
 Incubate at 42°C for 60 minutes
 Then, incubate at 70°C for 5 minutes to terminate the reaction.
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cDNA SYNTHESIS
Amplification of Single Strand cDNA by PCR
Mix the following components:
 2 μL of Reaction product
 5 μL of PCR Buffer
 1 μL dNTP mix
 3 μL MgCl2
 1,5 μL Forward Primer
 1,5 μL Reverse Primer
 0,5 μL Taq Polymerase
 35,5 μL Nuclease free Water
 Total volume is 50 μL.
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cDNA SYNTHESIS
Perform PCR in Thermal Cycle at following conditions:
First Denaturation:……. 94°C…………….3’
Denaturation …………..94°C……….……30’’
Annealing :………….... 58°C…………….30’’
35 cycle
Extention :……………. 72°C…………….45’’
Load PCR product into 1% agarose gel and perform electrophoresis. By using appropriate marker
compare and quantify the cDNA product.
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