I faced some problem in constructing a standard curve for my gene of interest. The GOI is somehow a low copy number genes. Therefore, I'm going to increase the starting amount of cDNA by 500 ng per tube reaction (which mean during DNase treatment , the starting total amount of RNA will be 5 ug).
I am doing qPCR using two step kit . I used iScript Reverse Transcription by Biorad to transcribed to the cDNA, but I am wondering whether I need to increase this reverse transcript volume as the volume of cDNA concentration will be increase to the total of 5 ug? I am still new to this real-time world.
Attached herewith is the protocol sheet for your reference. Thank you.
Thank you Nevin and Laurence. I did it and yes it is ok. I did test the reverse sample by conventional PCR and I get the expected band.
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