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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 |
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< | You can design your primers manually, or alternatively, we use this online tool from IDT. |
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> | You can design your primers manually, or alternatively, we use this online tool from IDT. |
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< | Preparation of DNA sample templates for QPCR |
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> | Preparation of DNA sample templates for QPCR |
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- Grow an overnight culture of each strain harboring your plasmid of interest in LB media supplemented with antibiotic
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- Grow an overnight culture of each strain harboring your plasmid of interest in LB media supplemented with antibiotic
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- 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
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- Extract total DNA from these pellets using a genomic DNA isolation kit
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- Extract total DNA from these pellets using a genomic DNA isolation kit
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Preparation of DNA for plasmid and standard curves |
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- For the gDNA standard curve, you will need to extract total DNA from wild-type E. coli strain not containing any plasmids
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- For the gDNA standard curve, you will need to extract total DNA from wild-type E. coli strain not containing any plasmids
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- It is generally good practice to use the same E. coli strain harboring your plasmid of interest
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- For the plasmid standard curve, you will need to mini-prep your plasmid
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- For the plasmid standard curve, you will need to mini-prep your plasmid
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QPCR using SYBR Green I dye, Part 1: Setting up a plate |
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- 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)
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- 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)
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- 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
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- Normalize your sample templates to 2ng/無
- 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
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- Normalize your sample templates to 2ng/無
- 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
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- For 96-well plates the reaction volumes are as follows:
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- Run you qPCR plate using the following cycling conditions:
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- Run your qPCR plate using the following cycling conditions:
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- Step 1 = Hold Stage
- 50蚓 - 02:00
- 95蚓 - 10:00
- Step 2 = PCR Stage
- 95蚓 - 00:15
- 54蚓 - 01:00
- Go to step 2-1, 40X
- Step 3 = Melt Curve
- 95蚓 - 00:15
- 54蚓 - 01:00
- 95蚓 - 00:15
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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
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-- DaciaLeon - 15 Dec 2016 |
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