Do AmpliTaq™ DNA Polymerase and AmpliTaq™ Gold™ DNA Polymerase add on extra A to the PCR product?

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Answer

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: E1077

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If I choose mixed bases, e.g., GC, for my oligo manufacturing, will it be a 50/50 mix?

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No, we do not guarantee 50/50 of mixed bases. If a mix of GC bases is requested, for example, the synthesizer would deliver half the normal amount of G and half the normal amount of C. Coupling efficiency is not taken into account. Therefore, it is possible that a mix, such as 30/70, will be delivered.

Answer Id: E7287

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I’m seeing smearing after PCR. What is causing this?

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Please see some reasons below for seeing smearing:

-The enzyme, primer, Mg2+, and/or dNTP concentration was too high.
-The annealing temperature was too low for the primers being used.
-Too many cycles were used.
-The annealing and extension times were too long.
-Bad or old primers.
-Too much template was used initially, try to start with 104-106 molecules
-Consider using additives or PCR Optimizer™ Kit (Cat. No. K122001), especially if you feel strongly that the primers should work/have worked before and are using Taq.

Answer Id: E7288

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Does the activation of AmpliTaq Gold™ DNA Polymerase at 95 degrees C for 10 min interfere with the half-life of the enzyme?

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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: E1078

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What are the unit definitions for AmpliTaq™ DNA Polymerase and AmpliTaq Gold™ DNA Polymerase?

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AmpliTaq™ DNA Polymerase: One unit (U) of enzyme is defined as the amount that will incorporate 10 nmoles of dNTPs into acid-insoluble material per 30 minutes in a 10-minute incubation at 74 degrees C under the analysis conditions specified in the product insert.

AmpliTaq Gold™ DNA Polymerase: One unit (U) of enzyme is defined as the amount that will incorporate 10 nmoles of dNTPs into acid-insoluble material per 30 minutes in a 10-minute incubation at 74 degrees C under the analysis conditions specified in the product insert.The enzyme is shipped in an inactive form. It is activated by heating for 3 hours at 80 degrees C before the activity is measured.

Answer Id: E2125

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I’m getting low yield of my desired fragment. What am I doing wrong and how can I increase my yield?

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Please see our suggestions below to increase yield:

-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR. [Lee (1995) BioTechniques 18:225].
-Not enough enzyme was used.
-Denaturation/extension temperature was too high and enzyme died prematurely.
-Too much DMSO (>10%).
-Incorrect annealing temperature: run a series of reactions using different annealing temperatures, starting 5 degrees below the calculated Tm.
-Too few cycles.
-Insufficient or too much Mg2+.
-Poorly designed primers: double check primer sequence against template sequence, primers should have similar melting temperatures, avoid complementary sequences at the 3’ end of primers.
-Carryover inhibitors (e.g., blood, serum).
-Denaturation time was too short. Genomic and viral DNA can require denaturation times of 10 minutes.
-Not a long enough extension time was used depending on the size of product being amplified.
-Use of super-irradiated (treated with >2500 mj/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR]. Alternatively, a combination of 1.0 M betaine with 6-8% DMSO or 5% DMSO with 1.2-1.8 M betaine can be used to amplify GC-rich templates [Baskaran (1996) Genome Res 6:633].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme, melanin, etc.). Add BSA to the PCR (~160-600 μg/mL), increase the amount of Taq, and/or increase the volume of the PCR to dilute out the inhibitor. The concentration of BSA to add may be dependent on the amount and type of inhibitor present. Additionally, fatty acid-free, alcohol-precipitated BSA, or Fraction V BSA all should be effective.

Answer Id: E7289

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What is the expected half life of AmpliTaq™ DNA Polymerase at 95 degrees C?

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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%.

Answer Id: E1139

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I’m getting no bands from my PCR product. What could cause this?

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Answer

Here are some reasons why your PCR experiment may be failing:

-NaCl at 50 mM will inhibit the enzyme.
-Too much KCl in the reaction. Do not exceed 50 mM.
-Incorrect annealing temperature was used.
-Incomplete denaturation (time and temperature must be long and high enough).
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR].
-10% DMSO partially inhibits Taq.
-Hemin (in blood samples) inhibits Taq.
-Use of super-irradiated (treated with >2500 mJ/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR [Lee (1995) BioTechniques 18:225].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme). Add BSA to the PCR, increase the amount of Taq, and/or increase the volume of the PCR to dilute out them inhibitor.

Answer Id: E7290

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What is the difference between AmpliTaq Gold™ polymerase and Platinum™ Taq polymerase?

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Answer

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.

Answer Id: E7267

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What does hot start PCR mean?

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Hot start is a way to prevent DNA amplification from occurring before you want it to. One way to do this is to set up the PCR reaction on ice, which prevents the DNA polymerase from being active. An easier method is a use a ‘hot-start’ enzyme, in which the DNA polymerase is provided in an inactive state until it undergoes a high-heat step.

Answer Id: E7270

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What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?

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Answer

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

Answer Id: E1083

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I’m getting an unexpected product when performing PCR. What could be the cause of this and what do you suggest I try?

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Answer

Please see the following possibilities and suggestions we have:

-Primer design: try longer primers to avoid binding at alternative sites, avoid 3 consecutive G or C nucleotides at the 3’ end.
-Annealing temperature: increase annealing temperature to increase specificity.
-Mg2+ concentration: try a lower concentration.
-DNA contamination: use aerosol tips and separate work area to avoid contamination, use UNG/UDG technique to prevent carryover.

Answer Id: E7291

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How much MgCl2 should be added to the PCR amplification when using AmpliTaq™ DNA Polymerase or AmpliTaq Gold™ DNA Polymerase?

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Answer

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: E1088

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How can I facilitate the amplification of templates with hairpin-loop structures or high GC-content?

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Answer

You can try adding 5-10% DMSO, up to 10% glycerol, or 1-2% formamide or a combination of these to facilitate difficult templates. Note: the use of cosolvents will lower the optimal annealing temperatures of your primers.

Answer Id: E7273

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How do I determine the percentage of full-length oligonucleotide?

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Answer

The percentage of full-length oligonucleotide depends on the coupling efficiency of the chemical synthesis. The average efficiency is close to 99%. To calculate the percentage of full-length oligonucleotide, use the formula: 0.99n-1. Therefore, 79% of the oligonucleotide molecules in the tube are 25-bases long; the rest are <25 bases. If you are concerned about starting with a preparation of oligonucleotide that is full-length you may want to consider cartridge, PAGE, or HPLC purification.

Answer Id: E7279

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