Description

KOD SYBR® qPCR Mix is a highly efficient 2x Master Mix for real-time PCR using SYBR® Green I and based on the 3'→5' exonuclease deficient KOD DNA Polymerase. The master mix contains all the required components, except the ROX reference dye and primers (50x ROX reference dye is supplied separately with this kit). The master mix aids reaction setup, and improves the reproducibility of experiments. This product was optimized to be highly efficient and robust in the SYBR® Green assay.

Features

  • Quantitative analysis can be achieved using long targets, up to 2kb.
  • Efficient for GC-rich targets
  • Effective amplification can be achieved using crude samples.

Applications

< KOD SYBR® qPCR Mix >
Intercalation assay with SYBR® Green I

Table 1.Compatible real-time instruments

Applied
Biosystems
ABI PRISM® 7000 Roche
Diagnostics
LightCycler® 1.x / 2.0
ABI PRISM® 7700 LightCycler® Nano
Applied Biosystems® 7300 LightCycler® 480
Applied Biosystems® 7500/7500FAST Bio-Rad / MJ MiniOpticon™
Applied Biosystems® 7900HT CFX96 Touch™
Applied Biosystems® StepOne™ Agilent Technologies Mx3000P / Mx3005P / Mx4000
Applied Biosystems® StepOnePlus™ TaKaRa Thermal Cycler Dice® Real Time Systems

Table 2.Comparison of properties with the conventional Master Mix

Conventional KOD SYBR® qPCR Mix
Enzyme Taq DNA KOD DNA Polymerase [exo(-) mutant]
Amplification Size 70~150 bp
(Maximum: 300 bp)
70 bp ~ 2 kb
High GC Targets Susceptible Not susceptible
Inhibition by impurities In crude samples Susceptible Not susceptible
(Suitable for amplification from crude specimens)

KOD SYBR® qPCR Mix was developed based on the unique properties (high efficiency, robustness) of KOD DNA Polymerase to enhance the convenience and versatility of the SYBR® Green I assay.

Storage condition

Store at -20ºC

Components

The reagent includes the following components for 40 reactions (QKD-201T) or 200 reactions (QKD-201), 50 µl total reaction volume:

<QKD-201T>

KOD SYBR® qPCR Mix 1 ml
50x ROX reference dye 50 µl

<QKD-201>

KOD SYBR® qPCR Mi 1.67 ml × 3
50x ROX reference dye 250 µl

Application data

Example 1.Enable the effective amplification of the targets, such as a high G/C(A/T) and/or a long target (up to 2 kb).

GC rich targets (GC content: >70%) were amplified using various real-time PCR master mixes. The targets were amplified successfully and quantitatively using KOD SYBR® qPCR Mix. [ABI StepOnePlus®]

  • KOD SYBR® qPCR Mix

  • High efficient master mix
    (Company A)

Target: IGF2R (189 bp / GC content: 83%)
Template: HeLa cDNA was synthesized using
Rever Tra Ace® qPCR RT Kit (Code No. FSQ-101)
with total RNA from HeLa cells.


A real-time PCR assay was performed using primers for conventional PCR. KOD SYBR® qPCR Mix exhibited quantitative amplification. [ABI StepOnePlus®]

Example 2.Enable the effective amplification from crude samples.

Genotyping of knock-in mice using mouse-tail lysates. [ABI 7500 Fast]
Primers were designed so that the amplicons were 100 bp (Tm: 79 ºC) and 341 bp (Tm: 84 ºC) for wild-type and knock-in, respectively. All genotypes were successfully detected.

Template: Mouse tail lysate (alkaline lysis method)
Primer ratio: F: WT: KI: = 0.2: 0.2: 0.67 mM (final)
Sample: Mouse tail lysate 2 µl / 20 µl reaction


One-tube ASP-PCR analysis using whole blood specimen. [ABI 7500 Fast]

SNP analysis was performed with a GC tailed primer from whole blood samples using KOD SYBR® qPCR Mix. All types of SNP were successfully determined by KOD SYBR® qPCR Mix. No signal was detected using the Taq-based conventional master mix (data not shown).

References

  • Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, and Imanaka T., Appl Environ Microbiol., 63: 4504-10 (1997)
  • Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y, J Mol Biol., 306: 469-77 (2001)
  • Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126: 762-8 (1999)