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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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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What does hot start PCR mean?

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Answer

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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The primers I am using worked for PCR initially, but over time, have stopped working. What happened?

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Answer

Primers should be aliquoted for single use before PCR set-up. Heat just the aliquoted primers to 94 degrees for 1 min. Quick chill the primer on ice before adding to the PCR reaction. Some primers may anneal to themselves or curl up on themselves.

Answer Id: E7302

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Do you have any resources to help design primers?

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Answer

Yes. OligoPerfect™ Designer can be used to design primers for sequencing, cloning, or detection.

Answer Id: E7276

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My oligonucleotide does not appear to be the right length when I checked by gel electrophoresis. Why is this?

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Answer

Oligos should be run on a polyacrylamide gel containing 7 M urea and loaded with a 50% formamide solution to avoid compressions and secondary structures. Oligos of the same length and different compositions can electrophorese differently. dC’s migrate fastest, followed by dA’s, dT’s, and then dG’s. Oligos containing N’s tend to run as a blurry band and generally have a problem with secondary structure.

Answer Id: E7303

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What are the minimum yield guarantees you offer for your oligos?

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Answer

The scale of synthesis is the starting point for synthesis, not the guaranteed final amount. We guarantee the total yield of oligonucleotide as a minimum number of OD units. Use this link (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html) for the minimum yield guarantees we offer for our oligos.

Answer Id: E7277

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

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Answer

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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Why is it difficult to amplify a GC-rich template?

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Answer

A GC-rich template often has a higher melting temperature and may not denature completely under the normal reaction conditions.

Answer Id: E7272

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

Product FAQ

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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How are these oligos quality controlled?

Product FAQ

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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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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Why are HPLC or cartridge purification not offered for larger oligos?

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Answer

As oligos increase in length, the column purification is less effective in separating the failure oligos from the correct products. PAGE purification would be the method of choice in this case.

Answer Id: E7283

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What are the melting temperatures for the M13 Forward (-20) and M13 Reverse primers in the TOPO™ Cloning and Zero Blunt™ Kits?

Product FAQ

Answer

Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.

Answer Id: E4024

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