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Product FAQ

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

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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Product FAQ

Why is coupling efficiency important?

Answer

Coupling efficiency is important as the effects are cumulative during DNA synthesis. The numbers below shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product available following synthesis of different length oligos. Even with a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can mean 15% more of the DNA present following synthesis is full-length product.

Number of bases added, 99% coupling full-length, Failures, 98% coupling full-length, Failures:
- 1, 99, 1, 98, 2
- 2, 98.01, 1.99, 96.04, 2.96
- 3,97.03, 2.97, 94.12, 5.88
- 10, 90.44, 9.56, 81.71, 18.29
- 20, 81.79, 18.21, 66.76, 33.24
- 30, 73.79, 26.03, 54.55, 63.58
- 50, 60.5, 39.5, 36.42, 63.58
- 95, 38.49, 61.51, 14.67, 85.33

Answer Id: E7278

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Product FAQ

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

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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Product FAQ

How many oligos do I need to order for a 96-well plate order or a 384-well plate order?

Answer

The plate orders must contain an average of 24 or more oligos per plate for 96-well plates or 192 or more oligos per plate for 384-well plates across the entire order.

Answer Id: E7285

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Product FAQ

What type of modifications does Life Technologies™ offer for my primers?

Answer

Please take a look at this list (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-modification-options.html) of standard modification options that we offer. If you do not see the modification option you would like, please email our Technical Support team at techsupport@thermofisher.com to see if we can accommodate your request.

Answer Id: E7280

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Product FAQ

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

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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Product FAQ

What is the fidelity of Platinum™ Pfx DNA polymerase (please quantitate)?

Answer

The rpsL fidelity assay was used to generate this data. Briefly, a plasmid containing an AmpR and SmS gene was amplified by PCR and religated. Transformations were plated on Amp plates and Amp/Sm plates. The mutant frequency = rpsL mutant colonies/total colonies x 100.
Results from this assay:
Taq: 4.8/100,000
Platinum™ Pfx: 1/1,000,000
Pfu were 1.5/1,000,000

Answer Id: E3026

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Product FAQ

How are these oligos quality controlled?

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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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

What does hot start PCR mean?

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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Product FAQ

How do I calculate the melting temperature of my primers?

Answer

A common equation used to calculate primer Tm is as follows: Tm (in degrees C) = 2 (A+ T) + 4 (G + C)

Answer Id: E7281

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Product FAQ

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

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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Product FAQ

Why doesn't Pfx DNA Polymerase yield the same quantitiy of PCR fragment as other thermostable proofreading polymerases?

Answer

Generally, it should. However, the 10X Pfx Amplification buffer is optimized to work with Platinum™ Pfx DNA Polymerase, so it is important Pfx is not used with buffers meant for other polymerases. Additionally, since the magnesium concentration is lower in the Pfx buffer, the primer annealing temperature may need to be lowered.

Answer Id: E3051

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