---+ 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 a plate 1 Set up standard curves for your gDNA and plasmid samples, these will also help you calculate your primer efficiencies (should be between 0.8-1.1) * 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, your CT values should be between 5-30 1 Normalize your sample templates to 2ng/µL * Make sure to dilute these so the concentrations fall within the range of the standard curves 1 Once your DNA templates have all been diluted, you can being to set up a 96- or 384- well plate to run your qPCR experiment * For 96-well plates the reaction volumes are as follows: %TABLE{dataalign="center"}% | *Template* | *10µM F primer* | *10µM R primer* | *SYBR Green PCR Mix* | *ddH<sub>2</sub>O* | | X | 0.75 | 0.75 | 7.5 | template - X | 1 Run your qPCR plate using the following cycling conditions: * Step 1 = Hold Stage 1 50°C - 02:00 1 95°C - 10:00 * Step 2 = PCR Stage 1 95°C - 00:15 1 54°C - 01:00 1 Go to step 2-1, 40X * Step 3 = Melt Curve 1 95°C - 00:15 1 54°C - 01:00 1 95°C - 00:15 ---++ QPCR using SYBR Green I dye, Part 2: Analyzing your data 1 Calculate the primer efficiencies by plotting CT vs DNA concentration for the gDNA and plasmid standards, and using [[https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/qpcr-efficiency-calculator.html][this website]] 1 If your efficiencies are between 0.8-1.1, then calculate the ratio of plasmid:gDNA for each of your samples -- Main.DaciaLeon - 15 Dec 2016
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Topic revision: r4 - 2017-02-23 - DaciaLeon