cDNA SYNTHESIS PROTOCOL

• First strand cDNA was synthesized using the ready to go T-primed first strand kit .
• For this, 2 μl of isolated total RNA was taken which was made up to 33 μl with DEPC-treated water in an RNase free microcentrifuge tube and heated for 5 minutes at 65ºC.
• The RNA solution was then incubated in a water bath set at 37 º c for 5 minutes. Simultaneously the first strand reaction mix was also incubated at 37ºC for 5 minutes.
• The RNA solution was then transferred to the first strand reaction mix. It was then incubated at 37ºc for 5 minutes. The contents of the tube were mixed properly by vortexing it gently.
• The tubes were then incubated for 90 minutes at 37oC. After the synthesis of first strand, the samples were stored at -20oC until further use.