*link will take you to our exclusive distribution partner site
Description
A convenient mastermix containing a new generation of hot-start polymerase that delivers improved specificity, yield, speed and robustness when amplifying targets from any template.
Product Highlights
Sensitive – incorporates MyTaq HS DNA Polymerase that exhibits increased affinity for DNA, thereby improving amplification of even limiting amounts of template
Efficient – novel buffer system maximizes efficiency of PCR amplification, delivering improved yield of any PCR product
Robust – reliable amplification in the presence of inhibitors and with even the most challenging DNA targets
Flexible – ideal for amplifying any target up to 5 kb from DNA extracted from human, animal and plant samples
Convenient – an all-in-one-tube mastermix that improves the speed, convenience and accuracy of PCR set-up
Fast – developed to give sensitive, reproducible and robust amplification of a broader range of targets under fast thermal cycling condition
Product Description
MyTaq™ HS Mix is recommended for PCR assays containing complex and low copy number targets as well as multiplex PCR. MyTaq HS Mix is comprised of MyTaq HS DNA Polymerase and a novel buffer system that deliver very high yield PCR amplification over a wide range of PCR templates. MyTaq HS has an increased affinity for DNA, enabling reliable amplification from even very low amounts of template. MyTaq HS has been developed to give more robust amplification than other commonly-used polymerases allowing it to perform well with challenging templates and in the presence of PCR inhibitors. Furthermore, the highly efficient nature of MyTaq HS means it gives excellent results under fast PCR conditions. MyTaq HS does not possess polymerase activity during the reaction set-up, thereby reducing the non-specific amplification that can hinder PCR assays from the start.
The product is supplied as a mastermix that requires the addition of only template, primers and water, thereby reducing the number of pipetting steps during PCR set-up for improved speed, throughput and assay reproducibility. The inclusion of dNTPs, MgCl2 and enhancers at optimal concentrations, helps eliminate the need for optimization, thereby saving time and ensuring comparable efficiencies for annealing and extension of all primers in the reaction, this makes MyTaq HS Mix ideal for multiplex PCR.
Applications
Fast PCR
Multiplex PCR
Genotyping
Complex templates (e.g. GC-rich)
Colony PCR
Low copy number PCR assays
High-throughput assays with prolonged PCR set-up
A 340 bp (A) and a 450 bp (B) fragment of the myc gene, a 525 bp (C) fragment of the EGFR gene and a 530 bp (D) fragment of the AGRI1 gene were amplified using MyTaq HS and hot-start DNA polymerases from Suppliers F, K, G and S. Each polymerase was used to set-up PCR reactions containing either 100 ng, 33 ng, 10 ng, 4 ng, 1 ng, 33 pg, 10 pg and 3 pg of human genomic DNA (Lanes 1-8 respectively), prepared by a 3-fold serial dilution. Marker is HyperLadder 1kb (M). MyTaq HS performed well across all four human genes.
M13 vector carrying a DNA insert was cloned into E.coli cells. 2 µL increments of agar (2a) and 2 µL increments of LB (2b) were added to a series of 50 µL PCR reactions (Lanes 1-8 respectively). The results show that MyTaq HS DNA Polymerase was more resistant to inhibition than that of supplier S. Individual colonies were picked from a plate of E.coli, washed directly into MyTaq buffer and amplified using MyTaq HS and primers for either a 2.6 kb or an 884 bp amplicon. Marker is HyperLadder 1kb (M). The results illustrate MyTaq is robust and gives highly–specific results.
Introduction to MyTaq
Overview, features and benefits of the MyTaq product family
All components are shipped on dry/blue ice and 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 of the reagents is retained until the expiry date on the outer box label.
Components may also be stored at +4°C if required, although storage at -20°C is recommended. When stored at +4°C, reagents will remain stable for a period of 2 weeks from date of receipt.
If possible, we would recommend an additional cleanup of affected samples, for instance, customers had very good experiences with the clean-up of samples containing humic acids with SureClean Plus. Alternatively decreasing the template concentration will help to minimize the amount of inhibitors, the amount of polymerase and primer can be increased, but do not exceed the suggested limits.
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.
During PCR setup at room temperature, standard polymerases will have some activity. When using a hot-start polymerase, this enzyme will have no activity at room temperature, thus reducing the risk of unspecific products in the reaction due to these mis-primed oligonucleotides. This feature makes this type of polymerase ideally suited to high-throughput applications, where the reactions may be left sitting at room temperature for prolonged periods of time.
All Meridian polymerases are available in the convenient format of a 2x master mix, containing all the reagents and additives necessary to perform successful PCR, with the exception of template, primers and water.
This formulation not only provides a convenient and hassle-free PCR setup, but also significantly reduces the chances of human error, inaccuracies and contamination by reducing the pipetting steps required.
We recommend a final reaction volume of 50 µL, this requiring the use of 25 µL of the 2x master mix and to make up to 50 µL using template, primers and PCR grade water.
Whilst the exact composition of the mixes is proprietary information, all mixes are made up of reaction buffer, magnesium, dNTPs, polymerase and additives, designed to keep the polymerase stable in the mix, as well as provide the optimal conditions for PCR.
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.
All Meridian PCR mixes come supplied at a 2x concentration, requiring the user simply to add template, primers and PCR grade water to a final concentration of 1x.
If possible, we would recommend an additional cleanup of affected samples, for instance, customers had very good experiences with the clean-up of samples containing humic acids with SureClean Plus. Alternatively decreasing the template concentration will help to minimize the amount of inhibitors. The amount of primer can be increased, but do not exceed the suggested limits.
