|
At Bioline 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 polymerase selection guide.
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.
As an alternative for quick and simple PCR optimization, we recommend using SureBand PCR Optimization kit.
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.
Please see the following table for the appropriate error rates:
|
| BIOLASE, BIOLASE Red, IMMOLASE, Mango Taq, Taq DNA Polymerase |
1.1 x 10-4 base substitutions/bp (Tindall and Kunkel, 1988) |
| 2.4 x 10-5 frameshift mutations/bp (Tindall and Kunkel, 1988) |
| 2.1 x 10-4 errors/bp (Keohavang and Thilly, 1989) |
| 7.2 x 10-5 errors/bp (Ling et al., 1991) |
| 8.9 x 10-5 errors/bp (Cariello et al., 1991) |
| 2.0 x 10-5 errors/bp (Lundberg et al., 1991) |
| 1.1 x 10-4 errors/bp (Barnes, 1992) |
ACCUZYME, ACCUZYME Red, AccuSure |
1.6 x 10-6 errors/base (Lundberg et al., 1991). |
One unit is defined as the amount of enzyme that incorporates 10nmoles of dNTPs into acid- insoluble form in 30 minutes at 72°C.
SAHARA DNA Polymerase is a highly sensitive, hot-start blend of polymerases, which has been specially formulated for use with low copy or challenging templates.
Due to its high processivity and high sensitivity, SAHARA™ allows for PCR product detection with as little as 1ng of template. As this will vary depending on the source and nature of the template, a template titration would be recommended to ascertain the optimal level that should be used.
During PCR set-up at room temperature, standard polymerases will have some activity. Hot-start polymerases do not have any activity at room temperature, thus reducing the risk of unspecific products in the reaction due to the presence mis-primed oligonucleotides. This feature makes this type of polymerase ideally suited to high-throughput applications, where the reactions may be required to remain at room temperature for prolonged periods of time.
The enzyme blend produces a mixture of A’ overhangs and blunt ends such that the PCR products are suitable for subsequent incorporation into a TA or blunt-ended vectors.
For the generation of a higher proportion of sticky ends, we recommend an extension temperature of 72°C, the optimal for the non-proofreading component. For a higher proportion of blunt ends, we recommend the user to extend at 68°C, the optimal for the proofreading component.
.
|
