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FAQ |  | Polymerases FAQ
To see FAQs about a specific Polymerases, see links from the specific product's page.
Q1: Which thermophilic DNA polymerase should I use?
Q2: What should I take into consideration when designing a set of PCR-primers?
Q3: How can I facilitate the amplification of templates with hairpin-loop structures or high CG-content?
Q4: How important is the quality of my DNA template in long PCR?
Q5: What kind of reaction tubes are recommended?
Q6: What is two-temperature PCR?
Q7: What if my primer extension reaction yields no product or a smear?
Q8: What causes an occasional smear in a "negative control" with no template present?
Q9: Can primer extension products be kinased in the primer extension reaction mixture?
Q10: How can I improve blunt-end ligation efficiency of primer extension products?
Q11: What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?
Q12: What is touchdown PCR?
Q13: Which NEB DNA polymerases can incorporate fluorescently labeled nucleotides?
Q1: Which thermophilic DNA polymerase should I use?
A1: The choice of a DNA polymerase for use in PCR-based applications is highly dependent on the characteristics of the system as well as the desired result. For routine amplifications where cost per reaction and yield are the priorities, NEB's Taq DNA Polymerase (NEB #M0267, NEB #M0273) is the industry standard. For routine systems where a hot-start polymerase is required, DyNAzyme II Hot Start Polymerase (NEB #F-504) is a hot start enzyme which reduces non-specific amplifications and primer-dimer formations, allowing for room temperature reaction set up, and is available through NEB's collaboration with Finnzymes, OY. For cost-effective routine PCR where high fidelity products are necessary, NEB's Vent DNA Polymerase (NEB #M0254) is an ideal and proven choice. For long amplicons, DyNAzyme™ EXT DNA Polymerase (NEB #F-505) can amplify targets up to 40 kb. It is especially well suited to handle demanding reaction conditions without time consuming optimization. When the ultimate in precision, speed, and sensitivity are required, Phusion™ High-Fidelity DNA Polymerase (NEB #F-530) can amplify targets up to 25 kb with extremely high fidelity and robustness, making it the optimal choice for cloning. Both Phusion and DyNAzyme EXT can be used for GC rich templates or templates with high degrees of secondary structure. Both are sold separately or in PCR kits (NEB #F-553 and NEB #F-552) which inlcude a 2X master mix so the user only needs to add DNA template and primers.
with extremely high fidelity and robustness, making it the optimal choice for cloning. Both Phusion and DyNAzyme EXT can be used for GC rich templates or templates with high degrees of secondary structure. Both are sold separately or in PCR kits (NEB #F-553 and NEB #F-552) which inlcude a 2X master mix so the user only needs to add DNA template and primers.
Q2: What should I take into consideration when designing a set of PCR-primers?
A2: 1. Avoid complementarity between the primers to prevent primer-dimer formation.
2. Avoid inverted repeats (self-complementarity).
3. CG-content of the primer should be around 50%.
4. Avoid G's and C's at the 3'-end of the primers.
There are many computer programs which will help you to design a primer-pair.
Q3: How can I facilitate the amplification of templates with hairpin-loop structures or high CG-content?
A3: You can try 5-10% DMSO, up to 10% glycerol, 1-2% formamide or combinations of these. Note: The use of co-solvents will lower the optimal annealing temperatures of your primers (e.g. 5.5-6°C in 10% DMSO). You could also try 7-deaza-dGTP in conjuction with normal dGTP in order to destabilize difficult structures. Note: 7-deaza-dGTP attenuates the signal of ethidium bromide staining.
Q4: How important is the quality of my DNA template in long PCR?
A4: Template preparation becomes particularly important when performing longer amplifications (>15 kb). Therefore, it is recommended to check the length of the DNA by agarose gel electrophoresis.
Q5: What kind of reaction tubes are recommended?
A5: We recommend thin-walled tubes especially for long PCR.
Q6: What is two-temperature PCR?
A6: If the Tm of both of your primers is high enough (over 65°C), the annealing and the extension steps can be combined into a single step.
Q7: What if my primer extension reaction yields no product or a smear?
A7: * Follow recommended conditions
* Optimize annealing and extension temperatures
* Optimize Mg2+ level
* Optimize amount of polymerase
* Purify DNA template by phenol/chloroform extraction and alcohol precipitation
* Avoid high salt carry-over into primer extension reaction
* Make sure dNTP solution has not undergone hydrolysis - try fresh dNTPs
* Cosolvents such as 100 mg/ml BSA, 1-10% formamide or 1-10% DMSO may help with some primer: templates
Q8: What causes an occasional smear in a "negative control" with no template present?
A8: DNA polymerases with low Km values for DNA, especially those with proofreading exonuclease functions, can cause primer artifacts to form if the DNA polymerase cannot bind to its preferred substrate (a 3´ end of an annealed primer). This primer artifact possesses single-stranded and double-stranded regions, and can appear either as DNA barely migrating out of the gel well or as a smear originating at the gel well. Taq DNA Polymerase (NEB #M0267, NEB #M0273) and DyNAzyme II Hot Start DNA Polymerase (NEB #F-504) can minimize this artifact.
Q9: Can primer extension products be kinased in the primer extension reaction mixture?
A9: No, because the ammonium sulfate in the reaction buffer inhibits T4 Polynucleotide Kinase (NEB #M0201). Instead, gel purify your DNA or use a G25 spin column. In a 50 µl reaction of 1X Kinase Reaction Buffer use up to 200 pmol of 5´-hydroxyl DNA termini with 1 mM ATP. Heat to 70°C for 5 minutes, then chill on ice. Add 20 units T4 Polynucleotide Kinase (NEB #M0201) and incubate at 37°C for 30 minutes.
Q10: How can I improve blunt-end ligation efficiency of primer extension products?
A10: Vector should be treated with alkaline phosphatase and the insert should have a 5´ phosphate. Other possibilities to improve ligation include: lowering the ATP level in the ligation buffer, increasing the insert:vector ratio or trying our Quick Ligation Kit (NEB #M2200).
Q11: What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?
A11: DNA Polymerase I, Large (Klenow) Fragment (NEB #M0210) and T4 DNA Polymerase (NEB #M0203) are the best choices for this application. DNA Polymerase I, Large (Klenow) Fragment can be used at 25°C or room temperature, but T4 DNA Polymerase must be used at 12°C due to its robust exonuclease. Both work well in a wide variety of buffers. Vent DNA Polymerase (NEB #M0254) and Deep Vent DNA Polymerase (NEB #M0258) can also be used but ThermoPol buffer must be used, the reaction temperature is high, and the enzyme cannot be heat inactivated. Mung Bean Nuclease (NEB #M0250) will chew back 3' overhangs but the strong exonuclease activity combined with the lack of polymerase activity yield a lower percentage of blunt ends.
Q12: What is touchdown PCR?
A12: It is a method for increasing specificity of PCR reactions. Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced (e.g. 1-2°C /every second cycle). The initial annealing temperature should be several degrees above the estimated Tm of the primers. Annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing temperature.
Q13: Which NEB DNA polymerases can incorporate fluorescently labeled nucleotides?
A13: Taq DNA Polymerase (NEB #M0267, NEB #M0273) and Vent (exo-) DNA Polymerases (NEB #M0257) are the best choices for incorporating fluorescently labeled nucleotides (1).
(1) Anderson, J.P., Angerer, B. and Loeb, L.A. (2005) Biotechniques 38, 257-264.
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