HiDi Taq DNA polymerase

Product#: 9201
$112.00

Size:

  • 250 Units
  • 1000 Units
Availability:
Ships in 1-2 weeks

HiDi Taq DNA polymerase

Cat. No. 9201
Size 250 units / 1000 units

Store - 20°C

Description

HiDi Taq DNA polymerase is a highly selective DNA polymerase variant, specially evolved for all assays in which High Discrimination is required, for instance in allele-specific PCRs, primer extensions or methylation-specific PCRs. HiDi Taq DNA polymerase efficiently amplifies from primers that are matched at the 3'-end and discriminates primers that are mismatched. An aptamer-based hot-start formulation of the HiDi Taq DNA polymerase prevents false amplification. Temperatures above 50°-55°C cause the aptamer's secondary structure to melt and will set-free the polymerase. HiDi Taq variant has a 5'-3'-nuclease activity and therefore can be used for hydrolysis probe-based real-time PCRs.
Features
  • Optimized for the amplification of about 60 - 200 bp.
    •  For >500 bp, the addition of additional Magnesium (+ 0.5 - 1.5 mM) might be needed 
  • Can be used in Real-time PCR.
    •  For detection in fluorescent dye (e.g. GreenDye or SYBR Green), HiDi DNA polymerase is recommended.
    •  For detection in hydrolysis probes,HiDi Taq DNA polymerase is recommended.
Application
  • SNP-detection by allele-specific amplification (ASA) / Allele-specific PCR
  • Methylation specific PCR (MSP)
  • Mutation detection
  • Liquid biopsy
  • HLA genotyping
  • Multiplex PCR
Composition / Content of the Product
  • HiDi and HiDi Taq DNA polymerase : 5 U/µl solution
  • 10x reaction buffer.
Data
9001S_1.png 
Fig.1 
Up : SNP (rs72921001) is a mutant of human chromosome 11, whose C (WT ; Wild Type) is substituted by A (SNP, Mutant) position 6868417. 
Bottom : Allele specific primer design. C-allele specific primer detects WT and A-allese primer detects SNP.

9001S_Fig1.gif     9001S_Fig11.gif 
Fig.2 
By using each allele specific primer designed in Fig.1, allele-specific PCRs were performed from 1 ng/µl of HeLa gDNA in the presence of a realtime dye, indicating the amplification of the C-allele specific primer only.
The A-allele specific primer is discriminated, thus not amplified up to 50 cycles.
9001S_Fig3.gif 
Fig.3
PCR products were subsequently analysed on a 2.5% agarose gel. A specific product is visualized by ethidium bromide staining at the right amplicon length of 109 bp

 

Product Name

Size

Code

Storage

Maker

HiDi DNA polymerase

250 units

9001S

-20℃

MYP

1000 units

9001M

HiDi Taq DNA polymerase

250 units

9201S

-20℃

MYP

1000 units

9201M


Quality Control
PCR activity: HiDi DNA polymerase was tested for successful ASA performance detecting a genomic SNP (rs72921001) in HeLa genomic DNA. PCR products were subsequently analysed on a 2.5% agarose gel. A specific product is visualized by ethidium bromide staining at the right amplicon length of 109 bp for the matched primer. In the case of the mismatch primer no product formation is visible after 50 cycles. HiDi Taq DNA polymerase was succesfully tested for hydrolysis probe based real-time PCR. The product demonstrates linearity of amplification over a specified serial dilution of human genomic DNA.

DNA polymerase activity: HiDi DNA polymerase activity has been monitored and adjusted to a specific DNA polymerase activity using an artificial DNA template and a DNA primer.

Enzyme-concentration has been determined by protein-specific staining. Please inquire more information at info@mypols.de for the lot-specific concentration.

No contamination has been detected in standard test reactions.

Related Product 1 : HiDi 2x PCR Master Mix
  • This does not include fluorescent dye. GreenDye, introduced below is recommended.
  • Cannot be used for detection by Hydrolysis Probes.

Product Name

Size

Code

Storage

Maker

HiDi 2x PCR Master Mix

100 tests

9101S

-20℃

MYP

500 tests

9101M

 
Related Product 2 : GreenDye 20x
GreenDye is a highly sensitive DNA binding dye suitable for qPCR applications and melting curve analysis.
GreenDye shows very low DNA polymerase inhibition when compared with other dyes in PCR.

It features a very high fluorescence when bound to double-stranded DNA and exhibits no degradation during PCR cycling.
It is a perfect dye for qPCR and high resolution melting applications.
  • Optimized for PCR experiments with HiDi DNA polymerase and HiDi Taq DNA polymerase
  • Can detect by regular SYBR/FAM filter channel.
  • Thermally stable for 50 cycles and more.
  • Suited for qPCR, melt curve analysis and High Resolution Melting (HRM)
  • No decrease in DNA polymerase performance at 1x concentration
9001S_Fig4.png 
Fig.4 GreenDye Melt Curve Analysis
All three dyes show a single peak indicating a single amplification product. GreenDye however, shows the sharpest melting peak.

Product Name

Size

Code

Storage

Maker

GreenDye 20× (200reactions)

250 μl

2000S

-20℃

MYP

GreenDye 20× (800reactions)

1000 μl

2000M


Material Safety Data (MSDS)
According to OSHA 29CFR1910.1200, Australia [NOHSC:1005, 1008 (1999)] and the EU Directives 67/548/EC, 1999/45/EC and 1272/2008 (CLP Regulation) any products which do not contain more than 1% of a component classified as dangerous or hazardous nor more than 0.1% of a component classified as carcinogenic, do not require a MSDS. However, we recommend the use of gloves, lab coats and eye protection when working with these or any other chemical reagents. myPOLS Biotec takes no liability for damage resulting from handling or contact with this product. This product is not hazardous, not toxic, not IATA-restricted. Product is not from human, animal or plant origin. The source of the product is recombinant protein expression in E. coli. The product is for research use only and may be used for in-vitro experiments only.

Important notes
  • Keep all components on ice.
  • Spin down and mix all solutions carefully before use.
  • HiDi 10x buffer is optimized for short amplicon length (about 60-200 bp, but also longer amplicon lengths are possible. The addition of additional Magnesium (+ 0.5 - 1.5 mM) might be needed in case of longer amplicons >500 bp. 
  • HiDi Taq DNA polymerase has a 5’-3’-nuclease activity and therefore can be used for hydrolysis probe-based assays.
  • HiDi Taq DNA polymerase is not suitable for real-time PCRs using a real-time dye such as SYBR Green. In this case, HiDi DNA polymerase (#9001) is recommended.
Licences/Patents/Disclaimers
This product is covered by a pending patent application. It is for the purchaser’s own internal research use and may not be resold, modified or used for production and commercial purposes of any kind without an agreement with myPOLS Biotec. For information on obtaining additional rights, please contact: info@mypols.de.

Recommendations for PCR/ Reaction Setup
PCR Mix

Component

Volume

Final concentration

Primer forward (10 µM)*

1 µl

0.2 µM (0.05- 1 µM)

Primer reverse (10 µM)*

1 µl

0.2 µM (0.05- 1 µM)

dNTPs (2 mM)

5 µl

400 µM

HiDi buffer (10x)

5 µl

1 x

HiDi Taq polymerase 5 U/µl

0.5 µl

2.5 U/reaction

Template/Sample extract

x µl

 

Nuclease-free  water

up to 25 µl total reaction volume

* Primers should ideally have a GC content of 40-60% typically
 
Typical 3-step PCR protocol

Initial denaturation

95°C

2 min

Denaturation

95°C

15 sec (25-40 cycles)

Annealing *

54–72°C

10 sec  (25-40 cycles)

Extension

72°C

30 sec/ 250 bp (25-40 cycles)

Hold

<10°C

 

* Typically, the annealing temperature is about 3-5°C below the calculated melting temperature of the primers used.

NOTICE THIS PRODUCT IS FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC USE.

References
  1. HiDi Taq DNA polymerase is based on: Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification. PLoS ONE 2014; 9(5): e96640. M. Drum, R. Kranaster, C. Ewald, R. Blasczyk, and A. Marx.
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