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SimpliFi HS Mix

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

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SimpliFi HS Mix

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

Fidelity comparison across commercially available high-fidelity polymerases.

The fidelity of the SimpliFi HS Mix was determined using a method designed by Lee D. et al. (Mapping DNA polymerase errors by single-molecule sequencing) using next generation sequencing and compared to other commercially available high-fidelity master mixes, using the same method. The results illustrate the higher fidelity (130x) of SimpliFi HS Mix.

GC bias with both high and low GC content

GC bias plot from whole-genome sequencing of a mixture of S. aureus and R. spheroides genomic DNA using SimpliFi HS Mix and a high fidelity mix from Supplier K. GC bias plots were generated, with %GC content of 100 bp windows on the X axis. Normalized coverage is indicated for the mixes and can be compared to a PCR-free reference library (representing unbiased coverage). The results illustrate SimpliFi HS Mix has a lower bias across the entire range of GC content, compared to the PCR-amplified library from supplier K, especially for GC percentages higher than 65%.

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
  • Blunt end cloning

Product Specification

Presentation BIO-25060: 100 x 50 µL Reactions: 2 x 1.25 mL
BIO-25061: 500 x 50 µL Reactions: 10 x 1.25 mL
Appearance Clear, colorless solutions
Hot start Aptamer mediated
Application Gene expression, Viral and bacterial detection, Robust PCR, High-specificity PCR, High-fidelity PCR, GC/AT-rich PCR, Blunt end cloning
Sample type DNA, cDNA
Presentation 2 vials / 10 vials
Storage -20 °C
Mix stability See outer label
Specific Activity 2x
Functional Single distinct bands of PCR product on an agarose gel and sensitivity
DNA Contamination No detectable product in a qPCR assay
DNase Contamination No detectable degradation

Product Ordering

Cat. No.              Size

BIO-25060          100 x 50µl Reactions

BIO-25061          500 x 50µl Reactions

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FAQs:

SimpliFi HS Mix

My DNA sample contains PCR inhibitors, which impairs my PCR results. What can I do for optimization?
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.
How long a fragment will this polymerase amplify?
SimpliFi HS Mix has been validated for templates up to 5 kb.
Which polymerase do I need for my specific application?
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.
What are the storage conditions and stabilities of Meridian polymerases?
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.
I am having problems optimizing my PCR, what would you recommend?
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.
What do the terms yield, fidelity, processivity and specificity actually mean?
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.
What is a proofreading polymerase?
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.
How is one unit of activity of a polymerase defined?
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.
What would be the recommended DNA polymerase for difficult samples like GC rich or bisulfite converted DNA?
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.
Is a sample available?
Please click here in order to request your sample. You will receive an email confirmation within two business days with delivery details.

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