Does AmpliTaq® DNA Polymerase have reverse transcriptase activity?
Does AmpliTaq® DNA Polymerase have exonuclease activities?
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.
Answer Id: 1073
Will PCR amplifications previously successful with regular AmpliTaq® Polymerase work as successfully with AmpliTaq Gold® Polymerase?
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.
Answer Id: 1074
Can AmpliTaq Gold® Polymerase be used in a one-step or a two-step RT-PCR?
Yes. We have the GeneAmp® Gold RNA PCR Reagent Kit (Cat. No. 4308206), which comes with a pAW109 kit control, as well as the GeneAmp® Gold RNA PCR Core Kit (Cat. No. 4312765) that does not contain the control reagents. For more information on how each kit can be used in either a one-step or two-step RT-PCR reaction, please refer to the GeneAmp® Gold RNA PCR Reagent Kit Protocol.
Answer Id: 1075
Why is AmpliTaq Gold® Polymerase the enzyme of choice for multiplex PCR?
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.
Answer Id: 1076
Do AmpliTaq® DNA Polymerase and AmpliTaq® Gold® DNA Polymerase add on extra A to the PCR product?
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.
Answer Id: 1077
Does the activation of AmpliTaq Gold® DNA Polymerase at 95 degrees C for 10 min interfere with the half-life of the enzyme?
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%.
Answer Id: 1078
Does AmpliTaq Gold® DNA Polymerase remain in an active state once it is activated?
What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?
Successful amplification of long PCR targets is dependent 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. Routine amplification of targets up to 5 kb has been successful with the use of AmpliTaq® Polymerase and AmpliTaq Gold® DNA Polymerase under standard PCR buffer conditions. However, for targets ranging from 5 kb up to 40 kb, rTth DNA Polymerase, XL works optimally. This enzyme and its optimized buffer promote not only efficient DNA synthesis, but also allow for correction of nucleotide misincorporations that might otherwise prematurely terminate synthesis. Please refer to the GeneAmp® XL PCR Kit Product Insert for more information.
Answer Id: 1083
When amplifying long PCR targets, is the concentration of the deoxynucleoside triphosphates (dNTPs) limiting?
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.
Answer Id: 1084
How much AmpliTaq® DNA Polymerase is used in a PCR amplification?
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.
Answer Id: 1086
How much MgCl2 should be added to the PCR amplification when using AmpliTaq® DNA Polymerase or AmpliTaq Gold® DNA Polymerase?
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.
Answer Id: 1088
What is "Hot-start" PCR?
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.
Answer Id: 1098
Is there anything to prevent AmpliTaq Gold® DNA polymerase from extending from the 3’ end of a TaqMan® probe in a 5’ nuclease assay?
When using AmpliTaq Gold® DNA polymerase, under what conditions would one choose a two-temperature vs. three-temperature PCR?
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.
Answer Id: 1505