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

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: 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
  2. Normalize your sample templates to 2ng/L
    • Make sure to dilute these so the concentrations fall within the range of the standard curves
  3. 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:

Template 10M F primer 10M R primer SYBR Green PCR Mix ddH2O
X 0.75 0.75 7.5 template - X

  1. Run your qPCR plate using the following cycling conditions:
    • Step 1 = Hold Stage
      1. 50C - 02:00
      2. 95C - 10:00
    • Step 2 = PCR Stage
      1. 95C - 00:15
      2. 54C - 01:00
      3. Go to step 2-1, 40X
    • Step 3 = Melt Curve
      1. 95C - 00:15
      2. 54C - 01:00
      3. 95C - 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 this website
  2. 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 - 23 Feb 2017 - 20:32:53 - Main.DaciaLeon
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