Ordering
Cat. No.
Size
BIO-21098
250 Units
BIO-21099
500 Units
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.
|
Reagent |
250 Units |
500 Units |
|
VELOCITY DNA Polymerase |
125 µL |
250 µL |
|
5x Hi-Fi Buffer (contains 10 mM Mg2+) |
2 x 1.5 mL |
4 x 1.5 mL |
|
50 mM MgCl2 Solution |
1 x 1.2 mL |
1 x 1.2 mL |
|
DMSO |
1 x 1.25 mL |
1 x 1.25 mL |
2 u/µL
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.
On Dry Ice or Blue Ice.
VELOCITY has been validated for long templates and has been successfully used for amplifications of fragments up to 30 kb.
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.
| 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. |
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