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View additional product information for AmpliTaq Gold™ Fast PCR Master Mix - FAQs (4390941, 4390939)
20 product FAQs found
AmpliTaq Gold和Platinum Taq都是热启动酶，均支持在无冰的情况下在实验台上配制反应体系。AmpliTaq Gold是一种化学修饰的热启动酶，以非活性状态提供。在95°C下，该酶可在10分钟后完全热激活。Platinum Taq是一种抗体介导的热启动酶。抗Taq抗体会与酶结合并抑制酶的活性，直至PCR反应的热变性阶段（30秒到2分钟），该酶的活性才被激活。
Both AmpliTaq Gold and Platinum Taq are hot-start enzymes that allow you to set up your reactions on the benchtop without the need for ice. AmpliTaq Gold is a chemically-modified hot-start enzyme, provided in an inactive state. Heat activates the enzyme, with full activity after 10 min at 95 degrees C. Platinum Taq is an antibody-mediated hot-start enzyme. The anti-Taq antibodies bind and inactivate the enzyme, until the heat denaturation step of the PCR reaction (30 sec to 2 min), which activates the enzyme.
A two-temperature PCR is commonly used when the primer annealing temperatures are above 60 degrees C. Use a three-temperature PCR when the templates have high G+C content and/or secondary structure, or desired primer annealing temperatures are below 60 degrees C. Please consult the Product Insert for more information on the use of AmpliTaq Gold DNA polymerase.
Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.
AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp 10X PCR Buffer I and/or GeneAmp 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).
No, AmpliTaq Gold DNA polymerase does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.
The fidelity of this PCR enzyme is affected in two ways. First, AmpliTaq DNA Polymerase typically binds to and incorporates base analogs less efficiently than conventional dNTPs, which means that polymerase activity is lower in reactions that contain base analogs. Second, the analog may pair with more than one conventional complementary template base, so the analog may be incorporated at an increased level compared to conventional dNTPs. For the best fidelity, we recommend that base analogs are included at low concentrations in the reaction.
The half-life of AmpliTaq DNA Polymerase at 95 degrees C is 40 min. During PCR, the sample is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95 degrees C is approximately 100 cycles.
Example: AmpliTaq DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.
Hot-start is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. The major obstacle to obtaining highly sensitive and specific amplifications appears to be competing side reactions such as the amplification of non-target sequences (mis-priming) and primer oligomerization. In an otherwise optimized PCR amplification, most non-specific products can be attributed to pre-PCR mispriming. Mispriming can occur any time all components necessary for amplification are present at permissive temperatures (below optimal annealing temperature) such as during reaction set up. A hot start can be performed either manually or can be automated utilizing AmpliTaq Gold DNA Polymerase.
In the manual hot-start technique a key component necessary for amplification, such as the enzyme, is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers. Once this temperature is reached, the missing component is added and the PCR amplification is allowed to proceed. Because a key component was withheld from the reaction at permissive temperatures, competing side reactions are minimized and specific amplification occurs.
AmpliTaq Gold DNA Polymerase facilitates the automation of the hot start technique and decreases the potential for contamination. AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase. Once activated, AmpliTaq Gold DNA Polymerase performs just as AmpliTaq DNA Polymerase does. Since it is provided in its inactive form, it can be added to a reaction without the fear of pre-PCR misprimed primers being extended. Once all of the components for amplification have been added to a tube, the reaction is heated to 95C for 5 - 10 minutes. This incubation activates the enzyme and allows the reaction to proceed normally.
The standard starting point is a final concentration of 1.5 mM magnesium ion. Since each molecule of dNTP (total 0.8 mM per reaction at 200 µM each) binds a magnesium ion, 0.8 mM magnesium ions are unavailable for AmpliTaq DNA Polymerase to use; hence, 0.7 mM free magnesium ions will be available as a cofactor for Taq's polymerization activity. It is important to note that there are other substrates in PCR amplifications that can also bind free magnesium (such as primers and template) therefore, the magnesium ion concentration should be titrated in order to find the optimum concentration for each reaction.
Most PCR amplifications use 2.5 units of AmpliTaq DNA polymerase per 100 µL reaction. A 25 µL reaction would use about 0.6 units. However, the optimal unit concentration per reaction should be empirically determined, and often the less is better rule applies. Using too much AmpliTaq may result in non-specific amplification.
The concentration of dNTPs in a standard PCR amplification is 200 µM each, for a total of 800 µM. This total dNTP amount corresponds to 39 µg of dNTPs. This is a huge excess and, when generating long PCR fragments, is not a limiting factor during the PCR amplification, as the amount of target DNA generated is generally no more than 1 µg. More importantly, the reaction condition variables need to be monitored more closely in order for successful long PCR amplification to occur.
Successful amplification of long PCR targets is dependent on variables such as sufficient extension time during the PCR amplification, cosolvent addition, pH of the reaction buffer, salt concentration, primer design, use of a hot start, DNA sample integrity, and the enzyme's proofreading and polymerase activities. A few examples of our long PCR enzymes include our Elonagase enzyme mix that can be used for amplicons up to 30kb (blend of Taq and proofreading enzyme) or our Phire Hot Start II enzyme mix that can be used for amplicons up to 20 kb (Taq polymerase). Read more here: https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/long-fragment-pcr.html
Yes, once activated, AmpliTaq Gold DNA polymerase remains active. Lowering the temperature will not inactivate AmpliTaq Gold DNA polymerase.
The half-life of AmpliTaq Gold Polymerase at 95 degrees C is 40 minutes. This is with constant incubation at the described temperature. During PCR, the reaction is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold Polymerase at 95 degrees C is approximately 100 cycles.
Example: AmpliTaq Gold DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t1/2 is at least 35 minutes; (35-40 min). Therefore, 35 min/20 sec/cycle = 105 cycles. 105 PCR cycles reduces enzyme activity by 50%.
Both AmpliTaq Gold DNA polymerase and AmpliTaq DNA Polymerase lack proofreading activity, so they will often leave a 3'-overhang. The base most often left is a 3'-A, however, the extra base appears to be sequence dependent and one cannot always rely on the fact that even just a single base has been left. In many cases, this artifact has been useful with TA Cloning kits. In order to drive the reaction to the extra A state, a final extension time at 72°C should be increased to 15-30 minutes.
Multiplex PCR involves the coamplification of multiple amplicons in a single PCR. Since multiple sets of primers are being added to a single reaction, the potential for primer dimer formation as well as a general loss of specificity and a decreased yield of specific product exists. The ability to control the activation of AmpliTaq Gold Polymerase via hot start, so that the multiple primers do not have the possibility to react with themselves, has proven successful at alleviating these complications and dramatically increasing specific product yield.
They will work more successfully and reproducibly with AmpliTaq Gold Polymerase. Although AmpliTaq Gold Polymerase is the same exact enzyme as AmpliTaq Polymerase, the fact that the reaction is being driven towards high specificity and yield may require some modifications to previous conditions. For example, if the previous reaction was on the edge of optimization, magnesium chloride concentrations may need to be re-optimized or if previous reactions were being run in a pH suboptimal for AmpliTaq Gold Polymerase, reaction conditions and sample preparation protocols may need to be revisited. Activation time for AmpliTaq Gold Polymerase will also need to be determined empirically and is dependent on cycler type.
AmpliTaq DNA Polymerase lacks a 3' - 5' exonuclease activity. However, the enzyme does have a fork-like, structure-dependent polymerization-enhanced 5 ' - 3' nuclease activity. During the extension step of a PCR amplification, the enzyme will hydrolyze any blocking strand starting from its 5' end, replacing the lost material by extending the new chain.
Yes, AmpliTaq DNA Polymerase has been reported to exhibit reverse transcriptase activity, but it is such a low and inefficient activity that it is neither useful nor harmful to the RNA PCR experiments.