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

Design your primers as outlined here

Overview

You will determine plasmid copy number by:

  • creating a standard curve for gDNA copy number (copy number vs cycle threshold) by loading known amounts of gDNA into a qPCR reaction.
  • creating a standard curve for plasmid number (number of copies vs cycle threshold) by loading known amounts of plasmid DNA into a qPCR reaction.
  • determining the number of copies of plasmid and genome in your experimental samples, and consequently, the number of plasmids/genome ("copy number").

Preparation of gDNA and plasmid DNA for creating standard curves.

  • Grow overnight cultures of:
    • Your experimental strain, harboring the plasmid the copy number of which you wish to determine.
    • Your experimental strain, however, without the plasmid.
  • 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)
  • For each sample, pellet 1 ml of cells for 5 minutes at 3,000 RPM
  • For gDNA template; perform a genomic DNA extraction on the strains do NOT harbor your plasmid.
  • For plasmid DNA template: perform a mini prep on strains that DO harbor the 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: r5 - 25 May 2017 - 20:15:04 - Main.SimonDAlton
 
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