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Product FAQ

What is "Hot-start" PCR?

Answer

Hot-start is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. The major obstacle to obtaining highly sensitive and specific amplifications appears to be competing side reactions such as the amplification of non-target sequences (mis-priming) and primer oligomerization. In an otherwise optimized PCR amplification, most non-specific products can be attributed to pre-PCR mispriming. Mispriming can occur any time all components necessary for amplification are present at permissive temperatures (below optimal annealing temperature) such as during reaction set up. A hot start can be performed either manually or can be automated utilizing AmpliTaq Gold DNA Polymerase.

In the manual hot-start technique a key component necessary for amplification, such as the enzyme, is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers. Once this temperature is reached, the missing component is added and the PCR amplification is allowed to proceed. Because a key component was withheld from the reaction at permissive temperatures, competing side reactions are minimized and specific amplification occurs.

AmpliTaq Gold™ DNA Polymerase facilitates the automation of the hot start technique and decreases the potential for contamination. AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase. Once activated, AmpliTaq Gold™ DNA Polymerase performs just as AmpliTaq DNA Polymerase does. Since it is provided in its inactive form, it can be added to a reaction without the fear of pre-PCR misprimed primers being extended. Once all of the components for amplification have been added to a tube, the reaction is heated to 95C for 5 - 10 minutes. This incubation activates the enzyme and allows the reaction to proceed normally.

Answer Id: E1098

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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

Can I use a DNA polymerase mixture containing both Taq polymerase and a proofreading polymerase for TA Cloning™?

Answer

If you wish to use a polymerase mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess with a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs following PCR. See the vector product manuals for details.

Some examples of Taq blends that are compatible with TOPO™ TA Cloning™ are Platinum™ Taq DNA Polymerase High Fidelity and AccuPrime™ Taq DNA Polymerase High Fidelity.

Answer Id: E4062

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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

What are Value Oligos?

Answer

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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Product FAQ

Do Elongase™ and Platinum™ Taq High Fidelity enzymes leave a 3'-A overhang on the PCR product for subsequent cloning into a TOPO™ TA Cloning™ or original TA vectors? What about Platinum™ Pfx polymerase?

Answer

Elongase™ and Platinum™ Taq High Fidelity polymerase mixes do leave 3' A overhangs on a portion of the PCR products, however, the cloning efficiency is greatly reduced from that obtained with Taq polymerase alone. Platinum™ Pfx polymerase does not leave 3' A overhangs. Therefore, with all proofreading enzymes or enzyme mixes that contain proofreading polymerases, we recommend that you treat the PCR product with Taq at the end of the PCR reaction, prior to TA cloning. To do this, add 1 U of Taq to a 50 μL reaction and incubate at 68-72 degrees C for 15 min. Phenol extract and ethanol precipitate the product before TA cloning.

Additional notes: The cloning efficiency decreases with increasing size of PCR products. For larger PCR fragments, we recommend that you gel-purify the PCR product and screen several clones. PCR primers should be designed with a 5' G, since Taq leaves a 3' A overhang preferentially on DNA ending in C.
Reference: Hu (1993) DNA and Cell Biology 12:763.
TA Cloning reference: Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A., and Smith, L.M. (1991) BioTechnology 9, 657.

Answer Id: E3064

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Product FAQ

I received my primer order, but the yield is lower than the scale that I ordered. Why is this?

Answer

The scale that is ordered refers to the starting synthesis scale, or amount of starting material used to create your oligo. Based on purification and efficiency, you will receive less than the starting synthesis scale. However, we do have a minimum yield guarantee based on the starting synthesis scale which can be found here: 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.

Answer Id: E7293

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Product FAQ

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

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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Product FAQ

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

Answer

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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Product FAQ

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

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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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

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

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

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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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

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

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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