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Taq DNA Polymerase with ThermoPol Buffer > FAQ |  | Taq DNA Polymerase with ThermoPol Buffer FAQ
See the Polymerases FAQ also.
Q1: Can Taq DNA Polymerase be used in other buffers?
Q2: Why is the product a smear when visualized on an agarose gel?
Q3: Why is there no product when visualized on an agarose gel?
Q4: How should I set up a PCR reaction using Taq DNA Polymerase?
Q5: The product sequence doesn't completely match the expected sequence. How can this result be improved?
Q6: What type of DNA ends result from a primer extension reaction or a PCR reaction using Taq DNA Polymerase?
Q7: When should Taq DNA Polymerase be used in a primer extension reaction or for PCR?
Q8: Will the 5'→3' exonuclease activity of Taq DNA Polymerase degrade primers?
Q9: Can Taq DNA Polymerase be used for nick translation?
Q1: Can Taq DNA Polymerase be used in other buffers?
A1: NEB's Taq DNA Polymerase retains 100% activity in both the supplied ThermoPol Buffer and commonly supplied Taq DNA Polymerase buffers which use MgCl2 and lack some of the salts and detergents present in ThermoPol. ThermoPol produces superior results in certain primer extension applications, but users can feel confident that NEB Taq DNA Polymerase will work well in competitors' buffers. For the convenience of our customers, we also sell our own version of the commonly supplied buffer under the name Standard Buffer.
Q2: Why is the product a smear when visualized on an agarose gel?
A2: Taq DNA Polymerase has a half-life of 45 minutes at 94°C, therefore conditions for making long products (>5kb) can degrade the polymerase. In this case, increasing the amount of Taq DNA Polymerase in the reaction can help. Vent DNA Polymerase (NEB# M0254) and Vent (exo-) DNA Polymerase (NEB# M0257) are more thermostable polymerases. DyNAzyme™ EXT DNA Polymerase (NEB# F-505) can amplify targets up to 40 kb, and Phusion™ High-Fidelity DNA Polymerase (NEB# F-530),Phusion™ Hot Start High-Fidelity DNA Polymerase (NEB# F-540) and Phusion™ Flash High-Fidelity Master Mix (NEB# F-548) amplify targets up to 25 kb (37 kb is the longest achieved) with extremely high fidelity.
Q3: Why is there no product when visualized on an agarose gel?
A3: 1. The DNA template is too long: seeQ3.
2. The DNA template is GC rich and contains secondary structure. Use a more thermostable polymerase such as Vent DNA Polymerase (NEB# M0254) or Vent (exo-) DNA Polymerase (NEB# M0257) and higher temperature. Alternatively, DMSO may be added to the reaction to lower the melting temperature of GC rich nucleic acids. Phusion™ (NEB# F-530), Phusion™ Hot Start (NEB# F-540), Phusion™ Flash (NEB# F-548) and DyNAzyme™ EXT (NEB# F-505) DNA Polymerases are optimal for amplification of GC rich templates or templates with high degrees of secondary structure.
3. Primer concentration may be too low. It should be above 0.4 μM.
4. Try fresh nucleotides.
Q4: How should I set up a PCR reaction using Taq DNA Polymerase?
A4: The general guidelines are:
0.5-2.0 units Taq DNA Polymerase
200 μM each dNTP
0.2-0.5 μM each primer
2-50 pg plasmid or 50-500 ng genomic template
1 X Buffer (Standard Taq or ThermoPol)
Denature at 94°C
Extend 1 minute/kb
Q5: The product sequence doesn't completely match the expected sequence. How can this result be improved?
A5: Taq DNA Polymerase does not posess a proofreading function. To reduce the error rate use a polymerase that does, such as Vent DNA Polymerase (NEB# M0254) or Vent (exo-) DNA Polymerase (NEB# M0257). Phusion™ High-Fidelity DNA Polymerase (NEB# F-530) offers the highest fidelity of any commercial thermostable polymerase.
Q6: What type of DNA ends result from a primer extension reaction or a PCR reaction using Taq DNA Polymerase?
A6: Taq DNA Polymerase generates single base 3' adenine overhangs, which are suitable for use in TA cloning vectors. Ends can be blunted by purifying DNA after primer extension and treating with a proofreading polymerase such as DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210), T4 DNA Polymerase (NEB #M0203) or Vent DNA Polymerase (NEB# M0254).
Q7: When should Taq DNA Polymerase be used in a primer extension reaction or for PCR?
A7: It is an excellent polymerase choice for routine primer extensions throughout a wide range of template sources. When cost per reaction and yield are priorities, Taq DNA Polymerase is the industry standard. It is limited by its relatively low thermostability (half-life of 45 minutes at 94°C) and lack of a proofreading exonuclease domain. These characteristics make it a poor choice for work on long (>5 kb) extensions, on templates with a high degree of secondary structure, or in applications that strive to minimize error incorporation.
Q8: Will the 5'→3' exonuclease activity of Taq DNA Polymerase degrade primers?
A8: No. The exonuclease will only degrade double stranded DNA that it encounters while extending a DNA fragment. It will degrade a secondary primer if bound to the same strand (e.g. a mutagenesis primer).
Q9: Can Taq DNA Polymerase be used for nick translation?
A9: Yes. This type of exonuclease activity can be used for nick translation and is also a property of DNA Polymerase I (NEB# M0209).
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