KiCqStart™ Universal SYBR® Green qPCR Protocol

Background
Instrument Compatibility for Kicqstart^® Sybr^® Green Qpcr Readymix™
Supplies
Assay Guidelines
Reaction Setup
PCR Cycling Protocol

Background

KiCqStart^® SYBR^® Green qPCR ReadyMix™ is a 2X concentrated, ready-to-use reaction cocktail that contains all components, except primers and template, for real-time quantitative PCR (qPCR). This unique combination of proprietary buffer, stabilizers, and KiCqStart^® Taq DNA polymerase delivers maximum PCR efficiency, sensitivity, specificity and robust fluorescent signal using fast, or conventional cycling protocols with SYBR^® Green qPCR.

Highly specific amplification is crucial to successful qPCR with SYBR^® Green I dye technology because this dye binds to and detects any dsDNA generated during amplification. KiCqStart^® Taq DNA polymerase contains a proprietary mixture of monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step (> 48 hours at room temperature). Activation of the enzyme is instantaneous at 95 °C. Replication of fragments up to 200 bp is complete in less than 20s at 60 °C.

For additional information, please see the qPCR Technical Guide or SYBR^® Green I-based qPCR technical animation.

Instrument Compatibility for KiCqStart^® SYBR^® Green qPCR ReadyMix™

Different real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. KiCqStart^® SYBR^® Green qPCR ReadyMix™ does not contain an internal reference dye. Please consult the following table to find the optimal kit for your instrument platform.

KiCqStart® SYBR® Green qPCR ReadyMix™	Compatible Instruments
KCQS00	Bio-Rad CFX384™, Bio-Rad CFX96™, Bio-Rad MiniOpticon™, Bio-Rad/MJ Chromo4™, Bio-Rad/MJ Opticon 2, Bio-Rad/MJ Opticon®, Cepheid SmartCycler®, Eppendorf Mastercycler® ep realplex, 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
KCQS01	Applied Biosystems 7500, Applied Biosystems 7500 Fast, Applied Biosystems ViiA 7, Stratagene Mx3000P®, Stratagene Mx3005P™, Stratagene Mx4000™
KCQS02	Applied Biosystems 5700, 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™
KCQS03	BioRad iCycler iQ™, BioRad iQ™5, BioRad MyiQ™

Supplies

• KiCqStart^® SYBR^® Green qPCR ReadyMix™ (KCQS00, KCQS01, KCQS02 or KCQS03 – select appropriate reagent based upon qPCR instrument used)
• Forward and reverse primers diluted to working concentration (10µM working stocks are sufficient for most assays)
  • Order custom oligos here
  • Predesigned gene expression primers are also available for most model organisms (KiCqStart^® SYBR^® Green Primers, KSPQ12012)
• Sterile filter pipette tips
• Sterile 1.5 mL screw-top microcentrifuge tubes (such as CLS430909)
• PCR tubes, select tubes to match desired format:
  • Individual thin-walled 200 µL PCR tubes (Z374873 or P3114)
  • Plates
    • 96 well plates (Z374903)
    • 384 well plates (Z374911)
  • Plate seals
    • ThermalSeal RTS™ Sealing Films (Z734438)
    • ThermalSeal RT2RR™ film (Z722553)
• PCR grade water (W1754)

Assay Guidelines

• Primer Design:
  
  • Predesigned primers: We offer a panel of predesigned primers that have been optimized for use with the KiCqStart^® SYBR^® Readymixes. These KiCqStart^® Primers can be ordered under product number KSPQ12012.
  • Custom Primers:
    • The design of highly specific primers is the single most important parameter for successful real-time PCR with SYBR^® Green I dye. The use of computer aided primer design programs is encouraged in order to minimize the potential for internal secondary structure and complementation at 3’-ends within each primer and the primer pair. Custom primers can be ordered under product number OLIGO.
  • Amplicon Size: KiCqStart^® SYBR^® Green qPCR ReadyMix™ can readily amplify fragments between 400 and 500 bp; however, to take full advantage of fast cycling protocols, amplicon size should be limited to less than 150 bp.
  • Primer Concentration: Optimal results may require titration of primer concentration between 100 and 500 nM. A final concentration of 300 nM for each primer is effective for most reactions.

• Assay Setup:
  
  • Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision. Assemble the reaction cocktail with all required components except sample template (genomic DNA or cDNA) and dispense equal aliquots into separate reaction tubes. Add the DNA template to each reaction as the final step. Dilution of template samples to allow at least a 5 µL addition will improve assay precision.
  • Suggested input quantities of template are: cDNA corresponding to 1 pg to 100 ng of total RNA; 100 pg to 100 ng genomic DNA

Reaction Setup

Reagent	Volume for 20 μL reaction	Final Concentration
KiCqStart® SYBR® Green qPCR ReadyMix™ (2X)	10.0 µL	1X
Forward Primer	variable	100 – 500 nM (start with 300 nM)
Reverse Primer	variable	100 – 500 nM (start with 300 nM)
Nuclease-free water (W1754)	variable	Add to q.s. to 20 µL
Template	at least 5 μL	variable
Final Volume (µL)	20 µL

Final reaction volume may vary from 10 to 50 μL, scale all components proportionally.
After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube.

PCR Cycling Protocol

	Fast 2-Step Cycling	Fast 3-Step Cycling	Standard Cycling
Initial denaturation:	95 °C, 30 sec *	95 °C, 30 sec *	95 °C, 2-3 min *
PCR cycling (30-45 cycles):	95 °C, 3-5 sec	95 °C, 3-5 sec	95 °C, 10-15 sec
		55 to 65 ºC, 15 sec
Collect data at end of extension step	60 °C, 20-30 sec †	68 to 72 °C, 10 sec †	60 °C, 30-60 sec †
Melt Curve (dissociation stage)	Refer to instrument instructions (optional)

* Full activation of KiCqStart Taq DNA polymerase occurs within 1 second at 95 ºC; however, optimal initial denaturation time is template dependent and will affect qPCR efficiency and sensitivity. Amplification of genomic DNA or supercoiled plasmid DNA targets may require 5 to 10 min at 95 ºC to fully denature and fragment the template. Short double-stranded DNA template (PCR product) or single-stranded DNA template, may require as little as 1sec at 95 ºC. Use 30 sec at 95 ºC as a general starting point.

^† Extension time is dependent upon amplicon length and minimal data collection time requirement for your qPCR instrument. Some primer sets may require a 3-step cycling protocol for optimal performance. Optimal annealing temperature and time or primer concentration may need to be empirically determined for any given primer set and real-time instrument.