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. Once you have prepared genomic and plasmid DNA as below, verify by melt curve that your primers produce single products.

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.

• 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.
• Use the qubit to determine concentration of DNA in both samples.

QPCR

• General guidelines on qPCR here
• Set up standard curves for your gDNA and plasmid samples.
  • To generate standard curves, perform serial dilutions of your gDNA and plasmid templates in ten-fold increments, e.g. 1:10, 1:100, 1:1000 etc.
  • A total of 7 dilutions is enough to make a good standard curve, your CT values should be between 10-30 cycles on qPCR machine.
• Normalize your sample templates to 2ng/µL
  • Make sure to dilute these so the concentrations fall within the range of the standard curves

1. 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:

1. Run your qPCR plate using the following cycling conditions:
   • Step 1 = Hold Stage
     1. 50°C - 02:00
     2. 95°C - 10:00
   • Step 2 = PCR Stage
     1. 95°C - 00:15
     2. 54°C - 01:00
     3. Go to step 2-1, 40X
   • Step 3 = Melt Curve
     1. 95°C - 00:15
     2. 54°C - 01:00
     3. 95°C - 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

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. Once you have prepared genomic and plasmid DNA as below, verify by melt curve that your primers produce single products.

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.
• Use the qubit to determine concentration of DNA in both samples.

• • Make sure to dilute these so the concentrations fall within the range of the standard curves

1. 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:

1. Run your qPCR plate using the following cycling conditions:
   • Step 1 = Hold Stage
     1. 50°C - 02:00
     2. 95°C - 10:00
   • Step 2 = PCR Stage
     1. 95°C - 00:15
     2. 54°C - 01:00
     3. Go to step 2-1, 40X
   • Step 3 = Melt Curve
     1. 95°C - 00:15
     2. 54°C - 01:00
     3. 95°C - 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

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

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.

• • 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.

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/µL
   • 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:

1. Run your qPCR plate using the following cycling conditions:
   • Step 1 = Hold Stage
     1. 50°C - 02:00
     2. 95°C - 10:00
   • Step 2 = PCR Stage
     1. 95°C - 00:15
     2. 54°C - 01:00
     3. Go to step 2-1, 40X
   • Step 3 = Melt Curve
     1. 95°C - 00:15
     2. 54°C - 01:00
     3. 95°C - 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

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 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/µL
   • 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:

1. Run your qPCR plate using the following cycling conditions:
   • Step 1 = Hold Stage
     1. 50°C - 02:00
     2. 95°C - 10:00
   • Step 2 = PCR Stage
     1. 95°C - 00:15
     2. 54°C - 01:00
     3. Go to step 2-1, 40X
   • Step 3 = Melt Curve
     1. 95°C - 00:15
     2. 54°C - 01:00
     3. 95°C - 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

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 may want to start 3-5 replicate cultures for each strain

1. 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)
2. For each sample, pellet 1 ml of cells for 5 minutes at 3,000 RPM

• • Our lab uses this one

Preparation of DNA for plasmid and standard curves

• • It is generally good practice to use the same E. coli strain harboring your plasmid of interest

QPCR using SYBR Green I dye, Part 1: Setting up a plate

• • 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

• • For 96-well plates the reaction volumes are as follows:

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

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

-- Main.DaciaLeon - 15 Dec 2016

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

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

-- Main.DaciaLeon - 15 Dec 2016

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