| |
| META TOPICPARENT |
name="ProtocolList" |
QPCR for quantification"> 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 . |
|
<
< | QPCR"> Designing primers for QPCR |
>
> | Designing primers for qPCR |
| | |
|
<
< | You can design your primers manually, or alternatively, we use this online tool from IDT. |
>
> | Design your primers as outlined here |
| | |
|
<
< | Preparation of DNA sample templates for QPCR |
>
> | Overview |
| | |
|
<
< |
- Grow an overnight culture of each strain harboring your plasmid of interest in LB media supplemented with antibiotic
|
>
> | You will determine plasmid copy number by: |
|
<
< |
-
- You may want to start 3-5 replicate cultures for each strain
- 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
- Extract total DNA from these pellets using a genomic DNA isolation kit
|
| | |
|
<
< | Preparation of DNA for plasmid and standard curves |
>
> |
- 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").
|
| | |
|
<
< |
- 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
- For the plasmid standard curve, you will need to mini-prep your plasmid
|
>
> | 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
- 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
- Normalize your sample templates to 2ng/µL
- Make sure to dilute these so the concentrations fall within the range of the standard curves
- 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:
- Run your qPCR plate using the following cycling conditions:
- Step 1 = Hold Stage
- 50°C - 02:00
- 95°C - 10:00
- Step 2 = PCR Stage
- 95°C - 00:15
- 54°C - 01:00
- Go to step 2-1, 40X
- Step 3 = Melt Curve
- 95°C - 00:15
- 54°C - 01:00
- 95°C - 00:15
QPCR using SYBR Green I dye, Part 2: Analyzing your data
- Calculate the primer efficiencies by plotting CT vs DNA concentration for the gDNA and plasmid standards, and using this website
- If your efficiencies are between 0.8-1.1, then calculate the ratio of plasmid:gDNA for each of your samples
-- DaciaLeon - 15 Dec 2016 |
Copyright © 2008-2025 by the contributing authors. All material on this collaboration platform is the property of the contributing authors.