Absolute QPCR for quantification of plasmid copy number in E. coli

This protocol is based on methods described in Lee et al (2006), link to paper.

Designing primers for QPCR

You can design your primers manually, or alternatively, we use this 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
2. Dilute your saturated cultures 1:100 into fresh media and let grow for 2-3 hours until the cells reach a mid-exponential phase (OD₆₀₀ = ~0.4-0.6)
3. For each sample, pellet 1 ml of cells for 5 minutes at 3,000 RPM
4. Extract total DNA from these pellets using a genomic DNA isolation kit
   • Our lab uses 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
2. 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

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.
2. To the electrocompetent cells, add 1-3 μl of DNA (<100 ng of DNA).
3. Mix by gently flicking the tube containing the electrocompetent cell + DNA mixture. Let the mixture sit on ice for 1-10 minutes.
4. 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.
5. Place the cuvette in the pulser and press the "Pulse" button.
   • For the TOP10 Electrocomp™ E. coli cells, the Ecl setting is fine.
6. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
7. Transfer the mixture to a 1.5 mL microcentrifuge tube.
8. 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.
9. Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
10. Incubate overnight at 37°C or other appropriate temperature.

-- Main.DaciaLeon - 15 Dec 2016