(r1) Barrick Lab :: QPCRToQuantifyPlasmidCopyNumber

---+ 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 

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---++ QPCR using SYBR Green I dye, Part 2: Running your samples

This procedure uses the TOP10 Electrocomp&trade; _E. coli_ cells.
   
   1 Thaw the electrocompetent cells on ice.
   1 To the electrocompetent cells, add 1-3 &mu;l 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&trade; _E. coli_ cells, the Ecl setting is fine.
   1 After electroporation, add 500-1000 &mu;l 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&deg;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 &mu;l) on an LB plate containing the appropriate antibiotic.
   1 Incubate overnight at 37&deg;C or other appropriate temperature.






-- Main.DaciaLeon - 15 Dec 2016
Barrick Lab > ProtocolList > QPCRToQuantifyPlasmidCopyNumber
Contributors to this topic
DaciaLeon, SimonDAlton, DanielDeatherage
Topic revision: r1 - 2016-12-15 - 21:22:57 - Main.DaciaLeon