 experiment, we will learn how to synthesize cDNA using RNA as template in molecular biology lab. After the RNA extraction, our next step is to synthesize cDNA because cDNA serves as a template in variety of downstream applications in RNA studies such as gene expression analysis. So synthesis of cDNA is usually the first step for various protocols in the molecular biology lab. We can synthesize cDNA in the lab by using a simple PCR technique we call as reverse transcriptase PCR or simply RT-PCR using an enzyme reverse transcriptase that is basically an RNA dependent DNA polymerase enzyme isolated from the retroviruses. So let's see that how to perform this experiment stepwise in the lab. For an ideal cDNA synthesis we need RNAs free environment. So the practices that need to follow to prevent RNA degradation and for optimal cDNA synthesis, we need to wear the powder free gloves to use the nucleus free reagents and lab wear and decontamination of your working area. For this experiment, commercially available reagents and enzymes are used. These reagents are extracted RNA sample, random primer, nucleus free water, dNTP mix, reaction buffer, riboloc RNA inhibitor and reverse it, reverse transcriptase. This experiment is performed in two steps. For the first step, place the PCR tubes into the cooling rack. Label the PCR tubes accordingly. Cut the micropipette to 10 microlitre. Now open the lids of the PCR tubes. Take 10 microlitre of RNA sample and then transfer it into the PCR tubes. Discard the use tip into the waste box. Now set the micropipette to 1 microlitre. Take 1 microlitre of random primer and transfer it into the PCR tubes. Discard the use tip in the waste box. Add 1 microlitre of nucleus free water into the PCR tubes. Close the lids of the PCR tubes. Place the tubes in the centrifuge and spin the tubes for a few seconds so that any reagent attached to the wall settles down in the bottom of the tube. After centrifugation, put the tubes on the cooling rack. Next step is incubation at 65 degree centigrade for 5 minutes. Switch on the thermal cycler and set the required conditions in it. Place the PCR tubes in the thermal cycler for the first step and close the lid of the thermal cycler. After setting all the required conditions, start the incubation, take out the PCR tubes and immediately place the tubes on the cooling rack. For the second step, now add the remaining reagents in the same PCR tubes. Set the micropipette to 4 microlitre. Take 4 microlitre of reaction buffer and add it in the PCR tubes. Set the micropipette to 2 microlitre, 2 microlitre of dNTP mix and add it in the PCR tubes. Use tip in the waste box. Now set the micropipette to 1 microlitre. Take 1 microlitre of riboloc RNA's inhibitor and add it in the PCR tubes. RNA's inhibitor is used to protect RNA from the activity of RNA's. Discard the use tip in the waste box. Take 1 microlitre of reward 8 reverse transcriptase and add it in the PCR tube. Reverse transcriptase also known as RNA dependent DNA polymerase is a DNA polymerase enzyme that synthesizes DNA using RNA as a template. Discard the use tip in the waste box. Close the lids of the PCR tubes. Spin the PCR tubes in the centrifuge for a few seconds. The PCR tubes into the thermal cycler. PCR conditions as shown on the thermal cycler screen reaction and wait till the whole process is completed. Completion store amplified DNA at minus 20 degrees centigrade or below until further use. Dear students, this was all about cDNA synthesis using the RNA template. Now we will quantify our cDNA using the nano-drop and then we will store it at minus 80 degree for the further use.