MBIO 4612 Lecture Notes - Lecture 2: Sybr Green I, Isopropyl Alcohol, 28S Ribosomal Rna
9 Feb 2020
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Should be free of dna (via dnase) Should be free of pcr inhibitors (contaminants from purification steps: ethanol, phenol, isopropanol = most common. Oligos (dt) string of deoxythymine (ts) No dna signal (overlap from other exons) Directly sybr green (quality of primers is critical! Indirectly fluorescent probes (this is very specific due to the extra binding event) Negative control (no dna) checks reagents for contamination. No reverse transcriptase control detects signals from any contaminating dna. Positive control checks reagents + primers work: very important if trying to show the absence of a gene. Same copy number in all cells: medium copy number is best correction more accurate, high will saturate assay too quickly, low will not be picked up by assay. No alternate splicing region if gene is spliced then it wont be detected. Multiplexing = look for several different genes in 1 reaction. Fluorophore + quencher attached to probe dna q prevents f from fluorescing when bound.
