---+ 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: Measuring primer efficiency It is important to measure the efficiency of your primers before assaying copy number in your samples. Generally, your primer efficiencies should be between 0.8-1.1 1 1 ---++ QPCR using SYBR Green I dye, Part 2: Running your samples This procedure uses the TOP10 Electrocomp™ _E. coli_ cells. 1 Thaw the electrocompetent cells on ice. 1 To the electrocompetent cells, add 1-3 μl of DNA (<100 ng of DNA). 1 Mix by gently flicking the tube containing the electrocompetent cell + DNA mixture. Let the mixture sit on ice for 1-10 minutes. 1 Pipette the mixture into a chilled cuvette, making sure that the mixture is at the bottom of the cuvette by gently tapping the cuvette on a flat surface. * Be sure to wipe any condensation off the sides of the cuvette before electroporation. 1 Place the cuvette in the pulser and press the "Pulse" button. * For the TOP10 Electrocomp™ _E. coli_ cells, the Ecl setting is fine. 1 After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells. 1 Transfer the mixture to a 1.5 mL microcentrifuge tube. 1 Incubate for ~30-120 minutes at 37°C or other appropriate temperaturein a shaking incubator. * Be sure to place the tube on its side so the transformed cells will grow properly. 1 Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic. 1 Incubate overnight at 37°C or other appropriate temperature. -- Main.DaciaLeon - 15 Dec 2016
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Topic revision: r1 - 2016-12-15 - DaciaLeon