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View additional product information for Phusion™ High-Fidelity DNA Polymerases (2 U/μL) - FAQs (F534S, F530L, F530S, F-530XL, F534L)
9 product FAQs found
Phusion Hot Start II DNA polymerase concentration is optimized to give good results in most reactions. When the PCR reaction is set up according to the instructions, the final concentration of Phusion enzyme is 1 U in 50 µL reaction (0.4 U in 20 µL reaction).
Phusion DNA Polymerases generate blunt end products; therefore, blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product with e.g. Taq DNA Polymerase (Cat. No. EP0401). However, before adding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion DNA Polymerase is very strong. Any remaining Phusion DNA Polymerase will degrade the A overhangs, thus creating blunt ends again.
Yes it is possible, especially when amplifying smaller amplicons. Processivity of Phusion DNA Polymerases is 10 times that of Pfu. We recommend extension times of 15 s/kb for Phusion Flash PCR Master Mix. 15 s/kb is a conservative value that we can promise to work with almost any amplicon. In many cases, significantly shorter extension times (0-5 s/kb) can be used without compromising the yield. What separates Phusion Flash DNA Polymerase from other fast polymerases is that all steps in the PCR protocol can be shortened, including annealing and denaturation. This results in extremely fast protocols as compared with any other polymerase.
Yes, protocols optimized for Phusion DNA Polymerase can be applied to Phusion Hot Start II DNA Polymerase reactions.
No separate activation step is required since Phire and Phusion Hot Start II DNA Polymerases are inactivated by a reversibly bound, specific Affibody ligand that dissociates during initial denaturation.
DyNAzyme II DNA Polymerase can use dUTP, biotinylated dNTPs, 7-deaza-dGTP, digoxigenin-dUTP, bromo-dUTP, radiolabeled dNTPs and ITP. DyNAzyme EXT DNA Polymerase and Phusion DNA Polymerase cannot read dUTP-derivatives or dITP in the template strand so the use of these analogues is not recommended. Use Phusion U Hot Start DNA Polymerase for amplification of dUTP and dITP containing templates.
Please read through the following information regarding low molecular weight smears from a Phusion High-Fidelity DNA Polymerase reaction:
- Increase annealing temperature.
- Decrease extension time.
- Decrease enzyme concentration.
- Optimize Mg2+ concentration.
- Titrate template amount.
- Decrease primer concentration.
- Design new primers.
Please read through the following information regarding high molecular weight smears from a Phusion High-Fidelity DNA Polymerase reaction:
- Decrease enzyme concentration.
- Decrease extension time.
- Reduce the total number of cycles.
- Increase annealing temperature or try 2- step protocol.
- Vary denaturation temperature.
- Optimize Mg2+ concentration.
- Decrease primer concentration
Please read through the following information regarding low yield or no product from a Phusion High-Fidelity DNA Polymerase reaction:
- Repeat and make sure that there are no pipetting errors.
- Use fresh high-quality dNTPs.
- Do not use dNTP mix or primers that contain dUTP or dITP.
- Sample concentration may be too low. Use more template.
- Template DNA may be damaged. Use carefully purified template and make sure template is not fragmented.
- Increase extension time.
- Increase the number of cycles.
- Optimize annealing temperature.
- Optimize enzyme concentration.
- Titrate DMSO (2-8%) in the reaction.
- Denaturation temperature may be too low. Optimal denaturation temperature for most templates is 98 degrees C or higher.
- Optimize the denaturation time.
- Check the purity and concentration of the primers.
- Check primer design.
- Try using the alternative GC Buffer.