*link will take you to our exclusive distribution partner site
Description
A DNA polymerase with 3’ – 5’ proofreading exonuclease activity that delivers exceptional fidelity and outstanding PCR yield even under fast PCR conditions.
Product Highlights
Accurate – possesses 3’ – 5’ proofreading exonuclease activity that delivers an error rate of 4.4 x 10-7 for excellent PCR fidelity
Efficient – highly processive enzyme and advanced buffering system for increased PCR yield from even the most challenging templates
Fast – extension rate of 1 5s/kb for ≤5 kb amplicons giving increased yield under fast thermal cycling conditions
Robust – highly processive nature of the enzyme confers an increased tolerance to impurities and ability to amplify complex templates
Flexible – ideal for high-fidelity amplification of targets up to 10 kb from human, animal and plant DNA
Product Description
VELOCITY DNA Polymerase is recommended for high-fidelity PCR amplification. The enzyme possesses a 3’ – 5’ proofreading exonuclease activity that provides an exceptional error rate of 4.4 x 10-7 for highly accurate amplification from a very broad range of human, animal and plant targets. Furthermore, VELOCITY is a highly processive enzyme with extension rates as fast as 15 s/kb for targets up to 5 kb and 30 s/kb for targets up to 10 kb, thereby enabling a reduction in PCR turnaround times.
VELOCITY delivers exceptional fidelity with outstanding PCR yield even from low template concentrations. The increased processivity of VELOCITY, results in shorter extension times for fast PCR, increased product yield and the ability to amplify longer fragments. VELOCITY also offers robust and reliable product yields, even in assays where PCR conditions are challenging, including the presence of impurities or GC-rich targets. VELOCITY is a high-performance DNA polymerase, ideally suited for high-yield, fast PCR amplification of even long targets containing no mutations.
Applications
High-fidelity PCR
Site-directed mutagenesis
Blunt end cloning
Fast PCR
High-yield PCR
Amplification of challenging templates
Long PCR
High-fidelity VELOCITY DNA Polymerase (1 unit), 2.5 units of Taq (2.5 units) or 4 units of Pfu DNA Polymerase (4 units) were used to amplify a 400 bp fragment of the β-globin gene from 100 ng human genomic DNA. The reactions were run for 30, 25, 20, 18 and 15 cycles (lanes 1-5 respectively) in HyperLadder 1kb (M). The results demonstrate the superior efficiency and processivity of VELOCITY, which requires less enzyme and fewer cycles to give high product yields.
High-fidelity VELOCITY DNA Polymerase, an alternative high fidelity polymerase from Supplier P and Pfu were compared in terms of their ability to amplify a 728 bp fragment of 77% GC-rich DNA, a 724 bp fragment of 68% GC-rich DNA, a 723 bp fragment of 67% GC-rich DNA and a 788 bp fragment of 71% GC-rich DNA (Lanes 1-4 respectively). HyperLadder 100bp (M). The results demonstrate that VELOCITY is better able to cope with GC-rich templates, giving the highest product yields.
High-fidelity VELOCITY DNA Polymerase was used to amplify 9.0, 6.0 2.0, 1.5 and 0.6 kb fragments from 100 ng genomic DNA human genomic DNA (Lanes 1-5 respectively). Reactions were set up with 1 unit of VELOCITY in a 50 µL reaction. 10 µL of the completed PCR reaction were then run on a gel. HyperLadder 1kb (M). The results demonstrate that VELOCITY can generate long PCR products with high yield and no mutations.
I did a side-by-side comparison of VELOCITY with Phusion. I got almost twice as much product with VELOCITY as from Phusion after cleanup and Nanodrop.
Jennifer Page, University of California, San Francisco
Product Selection
Please refer to the PCR Selection Chart to confirm the recommended product for your PCR application.
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 of the reagents is retained until the expiry date indicated on the outer box label.
""I did a side-by-side comparison of VELOCITY with Phusion. I got almost ... "
Jennifer Page, University of California, San Francisco
""In comparison to other DNA polymerases on the market, Meridian’s are ... "
Dublin City University, Ireland
"In comparison to other DNA polymerases on the market, Meridian’s are of the best quality at the most reasonable prices. Switching from our conventional high-fidelity enzyme to Meridian’s VELOCITY enzyme fixed one of the biggest problems I came across in my PhD project."
Dublin City University, Ireland
"I did a side-by-side comparison of VELOCITY with Phusion. I got almost twice as much product with VELOCITY as from Phusion after cleanup and Nanodrop."
Jennifer Page, University of California, San Francisco
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.
Concerning the polymerase concentration we recommend a range of 0.25–2.0 Units of VELOCITY 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 Velocity. Alternatively, you could pre-assemble the VELOCITY 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.
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.
