BIOL309 Lecture Notes - Lecture 3: Reverse Transcriptase, Reverse Transcription Polymerase Chain Reaction, Intron
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Use a template that binds the primers for sure (ideally a cdna population that expressed the gene for sure or a cloned dna: negative: Leave out the template (tests for dna contamination) Use a cdna pool from a cell type or mutant that doesn"t express the gene (tests that the primers are sensitive) Leave reverse transcriptase out of the cdna reaction (test for genomic dna contamination) if there happen to be dna in mrna, then it would still amplify with the primers. So if we leave out reserve transcriptase, we can check if the mrna is contaminated. Design primers that produce a product that spans an intron so that if the product was copied from a genomic dna template the product would be larger (test for genomic. If there is genomic dna, the pcr product would be larger. If there is cdna the pcr product would be smaller.