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 from crude samples

Specifications

• 50µl reaction volume
• Store at -20 ^oC
• 2x  Master mix
• 50x ROX reference dye
• Sybr I

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 Mix 1.67 ml × 3
50x ROX reference dye 250 µl

Table 1. Compatible real-time instruments

Table 2. Comparison of properties with conventional Master Mix

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.

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

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

REAL-TIME PCR KITS

DiagnoCine offers TOYOBO^® Taq DNA Polymerases in the form of Master Mix (Probe), SYBR Green Master Mix (Plus), Thunderbird qPCR (Probe), and Thunderbird SYBR qPCR formats. All Taq DNA Polymerases will provide the highest specificity by Hot Start technology, excellent Passive reference, and fits well for glass capillaries for many purposes.

• Real-time PCR Master Mix is a probe assay type with the highest hot start specificity, fast routine PCR experiments, and works well for GC-rich regions. This is for routine short-length PCR targets and not suitable for the long target (>10kb length; KOD is best for long-range size PCR) PCR. It can use for crude samples but KOD is best for crude sample amplifications. This product's strongest feature is the high specificity and Probe master mix for routine end-point PCR experiments.

• SYBR Green Realtime PCR Master Mix (plus for a hot start) is not as specific as the other Taq polymerases (i.e. Probe type or Thunderbird SYBR are more specific) master mix but this brand provides an excellent passive reference and fits for glass capillary use. It is designed to work well for routine short-range PCR and light GC-rich regions and fairly clean crude samples.

• Thunderbird Probe qPCR Mix is a probe assay type with hot start specificity, good passive reference, and glass capillary type PCR experiments. The strength is the highest specificity with low noise bands for difficult PCR and routine experiments such as genotyping and colony screening. Promised for best specificity and fast PCR speed but not made for a long-range PCR (>10kb length; KOD is best for long amplicons more than 10kb). It can unwind the GC-rich region and crude samples can be used (KOD is the best for crude sample use). This type is best for all routine laboratory PCR with short-mid range amplicons, looking for fast PCR time but with the strongest specificity and not dealing with PCR noise bands.

• Thunderbird SYBR qPCR Mix is an SYBR assay type with a strong specificity but slightly less specific than Probe qPCR. It has passive reference and glass capillary compatibility. The product is designed for a routine PCR with short to mid-range amplicons for a short and fast PCR time. The best performance is expected with partially clean crude samples and a fair level of GC-rich complexity.

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