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 just received my primers and they look yellow. Can I still use them?

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Most of the time the color should not affect PCR or any other experimental application since typically it is caused by the iodine used in the synthesis. There are some exceptions, however. Brown oligos can also be caused by the primer being overdried, and if this is the case, the primer may not work.

Answer Id: E7298

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Does AmpliTaq™ Gold DNA Polymerase contain exonuclease (proofreading) activity?

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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.

Answer Id: E1338

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

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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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When amplifying long PCR targets, is the concentration of the deoxynucleoside triphosphates (dNTPs) limiting?

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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: E1084

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How does a two-temperature protocol work and when would you suggest using one?

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You may choose to do a two-temperature protocol when the annealing temperature is relatively high. In this case, you would combine the annealing and the elongation steps, i.e., both can occur together at a temperature >62 degrees C. The advantage of a two-temperature protocol is that it is considerably quicker in comparison to the conventional three-temperature protocol.

Answer Id: E7274

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There is a green color in my lyophilized oligo. Can I still use it?

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If an oligo appears green in color, this is most likely due to ink falling into the tube. The oligo should still be fully functional. The color can be removed by doing an ethanol precipitation.

Answer Id: E7299

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

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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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Can you suggest some guidelines that will help me design my PCR primers?

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These guidelines may be useful as you design your PCR primers:

- In general, a length of 18-30 nucleotides for primers is good.
- Try to make the melting temperature (Tm) of the primers between 65 degrees C and 75 degrees C, and within 5 degrees C of each other.
- If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
- Aim for the GC content to be between 40 and 60%, with the 3’ of a primer ending in C or G to promote binding.
- Typically, 3 to 4 nucleotides are added 5’ of the restriction enzyme site in the primer to allow for efficient cutting.
- Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
- Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
- Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
- If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
- If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
- If you are using the primers for a PCR reaction to be used in TOPO™ cloning, the primers should not have a phosphate modification.
Read more about primer design tips and tools at https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html.

Answer Id: E7275

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There is a ball-shaped pellet at the bottom of my oligo tube. What is this and can I still use my oligo?

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Answer

If the oligo was overheated, it will appear as a “ball”-shaped pellet attached to the bottom of the tube. This should not affect the quality of the oligo, and the oligo should be readily soluble in water.

Answer Id: E7300

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Is there anything to prevent AmpliTaq Gold™ DNA polymerase from extending from the 3’ end of a TaqMan™ probe in a 5’ nuclease assay?

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Answer

Yes. There is a phosphate group on the 3' end of all TaqMan™ probes that prevents such extension.

Answer Id: E1408

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

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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 are Value Oligos?

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Value Oligos are the most cost-effective and fastest way to order oligos. They are available for 5-40-mers, at a 25 or 50 nanomole scale, with a range of purification options to suit your needs, and are eligible for next-day delivery. The cost is calculated per oligo as opposed to per base. Value Oligos are not available with modifications. Value Oligos undergo the same QC standards as our standard oligos with the same manufacturing process.

Answer Id: E7282

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How much AmpliTaq™ DNA Polymerase is used in a PCR amplification?

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Answer

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: E1086

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I’m missing a nucleotide in the middle of my sequence. How could this happen?

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Answer

There are two possibilities that could occur in any round of extension when creating your primer:

1.The added base is not detritylated correctly, missing one base addition but allowing possible extension in the next round.
2.The trityl group was removed, but not coupled or capped correctly before addition of the next base, allowing the chain to continue.

Answer Id: E7296

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