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BIOLASE™ DNA Polymerase FAQ

  1. Which polymerase do I need for my specific application?
  2. What are the storage conditions and stabilities of Bioline polymerases?
  3. I am having problems optimizing my PCR, what would you recommend?
  4. What do the terms yield, fidelity, processivity and specificity actually mean?
  5. What is a proofreading polymerase?
  6. What are the error rates of Bioline’s DNA Polymerases?
  7. How is one unit of activity of a polymerase defined?
  8. What is the extension rate of BIOLASE and BIOLASE Red DNA Polymerase?
  9. Is the product obtained using BIOLASE and BIOLASE Red DNA Polymerase suitable for use in TA cloning?
  10. What is the maximum length BIOLASE and BIOLASE Red DNA Polymerase will amplify?
  11. What is the ideal extension temperature of BIOLASE and BIOLASE Red DNA Polymerase?
  12. Will the dye in BIOLASE Red interfere with any applications carried out post-PCR?
  13. What are the concentrations of BIOLASE and BIOLASE Red DNA Polymerase?

1. Which polymerase do I need for my specific application?
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.

2. What are the storage conditions and stabilities of Bioline 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.

3. 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.

4. 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.
5. 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.

6. What are the error rates of Bioline’s DNA Polymerases?
Please see the following table for the appropriate error rates:

BIOLASE, IMMOLASE, Mango Taq, MyTaq DNA Polymerase , Taq DNA Polymerase.  1.1 x 104 base substitutions/bp (Tindall and Kunkel, 1988)
 2.4 x 105 frameshift mutations/bp (Tindall and Kunkel, 1988)
 2.1 x 104 errors/bp (Keohavang and Thilly, 1989)
 7.2 x 105 errors/bp (Ling et al., 1991)
 8.9 x 105 errors/bp (Cariello et al., 1991)
 2.0 x 105 errors/bp (Lundberg et al., 1991)
 1.1 x 104 errors/bp (Barnes, 1992)
ACCUZYME  1.6 x 106 errors/base (Lundberg et al., 1991).
VELOCITY  4.4 x 10-7 errors/base (Frey & Suppmann, 1995).

7. How is one unit of activity of a polymerase defined?
One unit is defined as the amount of enzyme that incorporates 10nmoles of dNTPs into acid-insoluble form in 30 minutes at 72°C.

8. What is the extension rate of BIOLASE and BIOLASE Red DNA Polymerase?
BIOLASE DNA Polymerase is a fast enzyme, which will extend fragments at 15-30s/Kb, dependant on template amplified.

9. Is the product obtained using BIOLASE and BIOLASE Red DNA Polymerase suitable for use in TA cloning?
Yes, BIOLASE DNA Polymerase does not possess 3’ to 5’ exonuclease activity, and therefore leaves an A-overhang at the 3’ end, thus making the PCR product suitable for integration into TA cloning vectors.

10. What is the maximum length BIOLASE and BIOLASE Red DNA Polymerase will amplify?
BIOLASE will comfortably amplify fragments of up to 5Kb on genomic templates.

11. What is the ideal extension temperature of BIOLASE and BIOLASE Red DNA Polymerase?
BIOLASE DNA Polymerase will extend between 50-80°C, however optimal extension will occur at 72°C.

12. Will the dye in BIOLASE Red interfere with any applications carried out post-PCR?
No, the dye in BIOLASE Red is completely inert, and as such will not interfere with any downstream applications. If you are concerned about these dyes interfering in your specific applications, we would recommend the cleanup of your samples using Bioline's SureClean or SureClean Plus column-free PCR Clean-Up Solution.

13. What are the concentrations of BIOLASE and BIOLASE Red DNA Polymerase?
BIOLASE DNA Polymerase is supplied at 5u/µl, whereas BIOLASE Red DNA Polymerase is supplied at 1u/µl.
   
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