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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My primer has an extra inserted base. How could this happen?

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Answer

If detritylation occurs inappropriately and/or if the synthesizer has an error and delivers the wrong base, an extra inserted base can occur in your primer. Please contact techsupport@thermofisher.com for assistance.

Answer Id: E7297

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Does the fidelity of AmpliTaq™ DNA Polymerase change in the presence of base analogs?

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Answer

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.

Answer Id: E1335

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

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Answer

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

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Answer

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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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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How are these oligos quality controlled?

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Answer

For 25, 50, and 200 nmol desalted and cartridge-purified DNA oligos, there is 100% A260 analysis. Random samples of 25% of the oligos produced are tested by either capillary electrophoresis or mass spectrometry. DNA oligos that are desalted and ordered at 25 and 50 nmol scales also have 100% real-time digital trityl monitoring during analysis. Desalted DNA oligos ordered at 1 and 10 μmols, DNA oligos at any scale that are purified by HPLC and PAGE, the majority of the DNA oligos with 3’ and/or 5’ modifications, and RNA oligos have 100% A260 analysis and capillary electrophoresis or mass spectrometry.

Answer Id: E7286

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

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Answer

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

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Answer

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

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Answer

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 don’t see a pellet in my oligo tube order. Should I ask for a replacement?

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Answer

The drying method dries the primer in a thin layer along the sidewalls of the tube instead of the bottom, therefore a pellet is not always visible and should still be ready to use.

Answer Id: E7301

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How does AmpliTaq Gold™ DNA Polymerase differ from AmpliTaq™ DNA Polymerase?

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Answer

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).

Answer Id: E1339

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I’m seeing high molecular weight EtBr stainable material left in wells. Why is this happening?

Product FAQ

Answer

This artifact occurs when either too many cycles were performed or too much DNA is added to the reaction. Try heating to 65 degrees C and putting sample on ice before loading.

Answer Id: E7292

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