SYBR® Green I Dye Quantitative PCR Protocol

Quantitative PCR Protocols

Although quantitative PCR uses the same basic concept as traditional PCR, the reactions differ in that the amplicons are generally smaller and are detected indirectly using an additional dye or labeled probe or primer.

In the PCR Technologies Guide, the requisite components and quality control requirements for qPCR experiments were described in detail. With those in mind, the following is a protocol that can be used as a basic template for qPCR incorporating SYBR Green I DNA binding dye that is amenable to modification and applicable for use as validation for a set of primers. Optimization of primer concentration and annealing temperature using SYBR Green I dye are described in further detail in Primer Concentration Optimization and Primer Optimization Using Temperature Gradient, respectively.

Equipment

  • Pipettes dispensing volumes from <1 to 200 μL
  • Benchtop microcentrifuge
  • Quantitative PCR instrument
  • Laminar flow hood for PCR set up (optional)

Supplies

Table P4-6A. SYBR Green PCR Mix Selection Guide.

Hot Start ReadyMixes (Taq, Buffer, dNTPs, Reference Dye, MgCl2)
KiCqStart® SYBR® Green qPCR ReadyMix™,
Cat. No. KCQS00
KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox ,
Cat. No. KCQS01
KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX,
Cat. No. KCQS02
KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ,
Cat. No. KCQS03
Compatible Instruments: Compatible Instruments: Compatible Instruments: Compatible Instruments:
Bio-Rad CFX384™ Applied Biosystems 7500 Applied Biosystems 5700 Bio-Rad iCycler iQ™
Bio-Rad CFX96™ Applied Biosystems 7500 Applied Biosystems 7000 Bio-Rad iQ™5
Bio-Rad MiniOpticon™ Fast Applied Biosystems ViiA 7 Applied Biosystems 7300 Bio-Rad MyiQ™
Bio-Rad MyiQ™ Stratagene Mx3000P® Applied Biosystems 7700  
Bio-Rad/MJ Chromo4™ Stratagene Mx3005P™ Applied Biosystems 7900  
Bio-Rad/MJ Opticon 2 Stratagene Mx4000™ Applied Biosystems 7900 HT Fast  
Bio-Rad/MJ Opticon®   Applied Biosystems 7900HT  
Cepheid SmartCycler®   Applied Biosystems StepOnePlus™  
Eppendorf Mastercycler® ep realplex   Applied Biosystems StepOne™  
Eppendorf Mastercycler® ep realplex2 s      
Illumina Eco qPCR      
Qiagen/Corbett Rotor-Gene® 3000      
Qiagen/Corbett Rotor-Gene® 6000      
Qiagen/Corbett Rotor-Gene® Q      
Roche LightCycler® 480      

 

Table P4-6B.

Hot Start ReadyMixes (Taq, Buffer, dNTPs, Reference Dye, MgCl2)
SYBR Green JumpStart Taq ReadyMix
for High Throughput qPCR
Cat. No. S9194
SYBR® Green JumpStart™ Taq ReadyMix™ for
Quantitative PCR, Capillary Formulation
Cat. No. S1816
Compatible Instruments: Compatible Instruments:
Applied Biosystems 5700 Roche LightCycler® 480
Applied Biosystems 7000  
Applied Biosystems 7300  
Applied Biosystems 7700  
Applied Biosystems 7900  
Applied Biosystems 7900 HT Fast  
Applied Biosystems 7900HT  
Applied Biosystems StepOnePlus™  
Applied Biosystems StepOne™  

 

Table P4-7. Reference Dye Concentration and Instrument Compatibility (when using qPCR reagents with a separate reference dye).

Instrument Final Reference
Dye Concentration
μL of Reference Dye
(per 20 μL Reaction)
Applied Biosystems 5700 0.2
Applied Biosystems 7000 0.2
Applied Biosystems 7300 0.2
Applied Biosystems 7500 0.1× 0.02
Applied Biosystems 7500 Fast 0.1× 0.02
Applied Biosystems 7700 0.2
Applied Biosystems 7900 0.2
Applied Biosystems 7900 HT Fast 0.2
Applied Biosystems 7900HT 0.2
Applied Biosystems StepOnePlus™ 0.2
Applied Biosystems StepOne™ 0.2
Applied Biosystems ViiA 7 0.1× 0.2
Bio-Rad CFX384™ not used -
Bio-Rad CFX96™ not used -
Bio-Rad MiniOpticon™ not used -
Bio-Rad/MJ Chromo4™ not used -
Bio-Rad/MJ Opticon 2 not used -
Bio-Rad/MJ Opticon™ not used -
Cepheid SmartCycler® not used -
Eppendorf Mastercycler® ep realplex not used -
Eppendorf Mastercycler® ep realplex2 s not used -
Illumina Eco qPCR not used -
Qiagen/Corbett Rotor-Gene® 3000 not used -
Qiagen/Corbett Rotor-Gene® 6000 not used -
Qiagen/Corbett Rotor-Gene® Q not used -
Roche LightCycler® 480 not used -
Stratagene Mx3000P® 0.1X 0.02
Stratagene Mx3005P™ 0.1X 0.02
Stratagene Mx4000™ 0.1X 0.02

 

Method

Assays run in KiCqStart ReadyMix are optimal when using a higher primer concentration than in conventional PCR. In the protocols below, 450 nM final concentration is used. This has been observed to be the optimal concentration for severalindependent assays. However some assays may benefit from further optimization and procedures for this are described in Primer Concentration Optimization and Primer Optimization Using Temperature Gradient.

1.    Place all reaction components on ice.

2.    Mix and briefly centrifuge to collect contents at the bottom of the tube.

3.    Prepare sufficient master mix to run all samples in duplicate.
       a. Include duplicate No Template Negative Controls (NTC).
       b. Calculate amount of reagents to mix. Add 10% volume to allow for pipetting error.
       c. Mix well, avoiding bubbles.

Table P4-8. Reaction Master Mix for KiCqStart Reagents.

Reagent Target Final
Concentration
Volume (μL) per
Single 20 μL Reaction
2× KiCqStart SYBR Green qPCR ReadyMix 10
Forward primer (10 μM stock) 0.45 μM 0.9
Reverse primer (10 μM stock) 0.45 μM 0.9
PCR grade water -
3.2

 

4.    Setup reactions:
       a. For NTC reactions, add 5 μL of water to the reaction tubes.
       b. For experimental reactions, add 5 μL of cDNA/gDNA solution to the reaction tubes.
       c. Visually confirm that all tubes or wells contain sample at the bottom at the correct volume.
       d. Carefully aliquot 15 μL of reaction master mix into each qPCR tube or plate well.
       e. Cap tubes or seal the PCR plate and label (according to instrument requirements). (Make sure the labeling does not
           obscure instrument excitation/detection light path.)
       f. Mix reactions well and spin if needed.

5.    Run samples according to the cycling protocol in Table P4‑9 and repeat the run of steps 1–3 for 40 cycles.
       (Note: These conditions are specific for FAST cycling protocols).

Table P4-9. Fast PCR Cycling Conditions.

FAST Cycling Conditions Temp (°C) Time (sec)
Initial Hot Start/denaturation 95 30
Steps 1–3 are repeated through 40 cycles
Step 1 95 5
Step 2
58 15
Step 3
72 10

Note: Use standard dissociation curve protocol (data collection)

 

6.    Refer to qPCR instrument manual and Data Analysis for guidance on data analysis.

Materials