---+ Absolute QPCR for quantification of plasmid copy number in _E. coli_ This protocol is based on methods described in Lee _et al_ (2006), [[http://www.sciencedirect.com/science/article/pii/S0168165605007509][link to paper]]. ---++ Designing primers for QPCR You can design your primers manually, or alternatively, we use this [[https://www.idtdna.com/Primerquest/Home/Index][online tool from IDT]]. ---++ Preparation of DNA sample templates for QPCR 1 Grow an overnight culture of each strain harboring your plasmid of interest in LB media supplemented with antibiotic * You may want to start 3-5 replicate cultures for each strain 1 Dilute your saturated cultures 1:100 into fresh media and let grow for 2-3 hours until the cells reach a mid-exponential phase (OD<sub>600</sub> = ~0.4-0.6) 1 For each sample, pellet 1 ml of cells for 5 minutes at 3,000 RPM 1 Extract total DNA from these pellets using a genomic DNA isolation kit * Our lab uses [[https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-extraction/genomic-dna-extraction/purelink-genomic-dna.html][this one]] ---++ Preparation of DNA for plasmid and standard curves 1 For the gDNA standard curve, you will need to extract total DNA from wild-type _E. coli_ strain not containing any plasmids * It is generally good practice to use the same _E. coli_ strain harboring your plasmid of interest 1 For the plasmid standard curve, you will need to mini-prep your plasmid ---++ QPCR using SYBR Green I dye, Part 1: Setting up the standard curves It is important to measure the efficiency of your primers, you can do this using your standard curves. Generally, your primer efficiencies should be between 0.8-1.1. 1 To generate standard curves, dilute your gDNA and plasmid templates in ten-fold increments * A total of 7 dilutions is enough to make a good standard curve * You might have to do this multiple times to get a curve in a good range, your CT values should range between 5-30 1 To generate standard curves, dilute your gDNA and plasmid templates in ten-fold increments ---++ QPCR using SYBR Green I dye, Part 2: Running your samples -- Main.DaciaLeon - 15 Dec 2016
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Topic revision: r2 - 2016-12-15 - DaciaLeon