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Ordering

Cat. No.
Size
BIO-25060
100 x 50µl Reactions
BIO-25061
500 x 50µl Reactions

*link will take you to our exclusive distribution partner site

Description

A high-fidelity polymerase mix using aptamer-based hot-start technology, highly suited to amplification of DNA from crude samples, for fast, inexpensive target enrichment, NGS library amplification and cloning applications.

Product Highlights

  • Robust - optimized enzyme/buffer mix promotes reliable amplification of a broad range of targets, including complex DNA extracted from human, animal and plant samples
  • Specific - the aptamer-based hot-start remains completely inactive during PCR set-up and after amplification, to prevent non-specific products
  • Optimized - high yields with minimal optimization, regardless of a template’s GC content, reducing time to results and eliminating the cost of unnecessary repeats
  • Enhanced accuracy - higher than 130x Taq fidelity, reducing errors for next generation sequencing (NGS) library amplification

Product Description

SimpliFi HS Mix is a combination of the latest advances in buffer chemistry and PCR enhancers and stabilizers, together with an aptamer-mediated hot-start polymerase, dNTPs and MgCl2. It has been designed for highly reproducible, accurate assay results in the presence of inhibitors. The advanced buffer chemistry and enhancers has been developed for fast PCR and is designed for superior sensitivity and specificity, making SimpliFi HS Mix perfect for NGS library amplification.

Applications

  • Gene expression
  • Viral and bacterial detection
  • Robust PCR
  • High-specificity PCR
  • High-fidelity PCR
  • GC/AT-rich PCR
Main

PCR Enzyme Guide

Download the PCR Enzyme Guide with detailed product descriptions and performance data to help you choose the best product for your research

PCR Selection Chart

Select the best reagent for your research

Product Selection

Please refer to the PCR Selection Chart to confirm the recommended product for your PCR application.


Specification

Components

Reagent

100 Reactions

500 Reactions

SimpliFi HS Mix

2 x 1.25 mL

10 x 1.25 mL

Concentration

  • 2x

Storage & Stability

All components should be stored at -20°C upon receipt for optimum stability. Repeated freeze/thaw cycles should be avoided.

When stored under the recommended conditions and handled correctly, full activity is retained until the expiry date indicated on the outer box label.

Shipping conditions

Shipped on Dry Ice or Blue Ice.



FAQs

SimpliFi HS Mix is designed to work in the presence of inhibitors. If there is too much interference the concentration of magnesium can be increased, for example, up to 4 mM (in the final reaction) should be added in presence of more than 10% of whole blood.

SimpliFi HS Mix has been validated for templates up to 5 kb.



Concerning the polymerase concentration we recommend a range of 0.25–2.0 Units of SimpliFi HS Mix in a 50 µL reaction and suggest to start with the lowest concentration. Do not to exceed 2 u (1 µL) in 50 µL reactions. Furthermore, we highly recommend the use of 3% DMSO for optimal polymerase performance. For difficult templates (genomic DNA, high-GC content, complex structural organisation) a higher concentration of DMSO could be advantageous. We would recommend doing a titration up to 10% DMSO, however in this case the annealing temperature should be reduced since DMSO decreases the melting point of primers by up to 5°C.



We do not have a readymade mix or buffer for SimpliFi HS Mix. Alternatively, you could pre-assemble the SimpliFi HS Mix buffer as a 2x or 3x buffer, adding all the reagents except for the primer and the enzyme, which cannot stay with the other reagents at 4 degrees for long. You will need to add the enzyme, template and primers before the reaction.



At Meridian we pride ourselves in supplying high-quality polymerases to suit your requirements. To aid your selection of the most suited enzyme for your specific applications, please see our enzyme selection tool.

All our polymerases are guaranteed for a period of 12 months from the date of purchase. These should be stored at -20°C during this time for optimal retention of activity.

Please Note: We do not recommend the storage of our polymerases at -80 °C as ice crystals could form on the active site, which may affect or destroy the activity of the enzyme.

PCR can be a challenging technique, with various parameters to optimize to achieve the best results. If you are having problems, these could be easily resolved by addressing a few issues. Please see our PCR troubleshooting guide for suggestions and help with your specific problems.

Observation Recommended Solution(s)
No or low PCR yield  Enzyme concentration too low – increase the amount of enzyme in 0.5 U increments.
 Primers degraded – check quality and age of the primers.
 Magnesium concentration too low – increase concentration in 0.25 mM increments with a starting  concentration of 1.75 mM.
 Primer concentration not optimized. Titrate primer concentration (0.3-1 µM); ensuring that both  primers have the same concentration.
 Template concentration too low – Increase concentration of template.
 Perform a positive control to ensure that the enzyme, dNTPs and buffers are not degraded and/or contaminated.
Multiple Bands  Primer annealing temperature too low. Increase annealing temperature. Primer annealing should be at least 5°C below the calculated Tm of primers.
 Prepare master mixes on ice or use a heat-activated polymerase.
 For problems with low specificity. Try adding 3% DMSO (not supplied) to improve specificity.
Smearing or artifacts  Template concentration too high. Prepare serial dilutions of template.
 Too many cycles. Reduce the cycle number by 3-5 to remove non-specific bands.
 Enzyme concentration too high - decrease the amount of enzyme in 0.5 U increments.
 Extension time too long. Reduce extension time in 0.5-1 minute increments.


These terms refer to parameters to be considered when performing PCR and are important features in selecting the correct enzyme for your needs. Understanding what they mean is therefore crucial: Yield: The amount of DNA produced in a PCR reaction. Fidelity: The accuracy of the enzyme at incorporating the correct dNTP to the elongating DNA strand. Processivity: The length of time a polymerase is associated with the template and therefore the size of fragment which can be amplified. Specificity: A measure of the unwanted by-products generated in a reaction.

In nature, the ability of a DNA polymerase to correct misincorporations of nucleotides in the DNA strand being elongated is often crucial to the survival of the host organism. This ability is termed "proofreading activity" and occurs in the 3' to 5' direction. This activity also leads to the polymerase removing unpaired nucleotides overhanging at the 3'end (A-overhangs), creating blunt ends.

One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acid-insoluble form in 30 minutes at 72°C.

The MyTaq HS DNA polymerase works very well with difficult samples like GC rich or bisulfite converted DNA. Especially the hot-start is important, as it decreases primer dimer and unspecific amplification. We would suggest to start with the recommended protocol. If optimization is needed, we would suggest to optimize the annealing temperature. Due to the degraded DNA after bisulfite conversion it may be useful to increase the amount of template and polymerase.

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