What PCR enzyme would you recommend for use with the Directional TOPO® Cloning Kits?

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For the Directional TOPO® Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50™ or Accuprime™ Pfx and Accuprime™ Pfx Supermix from Life Technologies are recommended for use.

When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO® reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.

Answer Id: 4290

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27584e8cefba0a67a8d1684d55a2a16a_FAQ

What is the difference between Platinum® technology and AccuPrime™ technology?

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With Platinum® technology, anti-DNA polymerase antibodies bind to the enzyme until the denaturing step at 94 degrees C, when the antibodies degrade. The polymerase is now active and primer extension can occur. AccuPrime™ Taq combines Platinum® Taq (Taq + Platinum® antibodies) with proprietary thermostable AccuPrime™ accessory proteins. The 10X reaction buffer contains the accessory proteins which enhance specific primer-template hybridization during each cycle of PCR.

Answer Id: 7266

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439fca360bc99c315c5882c4432ae7a4_FAQ

What are the main steps in PCR?

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The main steps are: denaturation, annealing, and extension. The template is typically heated to a high temperature (around 94-95 degrees C) allowing for the double-stranded DNA to denature into single strands. Next, the temperature is lowered to 50-65 degrees C, allowing primers to anneal to their complementary base-pair regions. The temperature is then increased to 72 degrees C, allowing for the polymerase to bind and synthesize a new strand of DNA.

Answer Id: 7269

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35d8f387d4934b6ee53ce5c9a1d8c1d7_FAQ

What does hot start PCR mean?

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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: 7270

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7f489f642a0ddb10272b5c31057f0663_FAQ

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

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

Answer Id: 7272

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d2a1e34d86293cb12f959f89dddf263e_FAQ

How can I facilitate the amplification of templates with hairpin-loop structures or high GC-content?

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You can try adding 5-10% DMSO, up to 10% glycerol, or 1-2% formamide or a combination of these to facilitate difficult templates. Note: the use of cosolvents will lower the optimal annealing temperatures of your primers.

Answer Id: 7273

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9d702ffd99ad9c70ac37e506facc8c38_FAQ

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: 7274

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1301962d8b7bd03fffaa27119aa7fc2b_FAQ

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.lifetechnologies.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html.

Answer Id: 7275

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bb921944c8c4531826da3fa99b494c1a_FAQ

Do you have any resources to help design primers?

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

Answer Id: 7276

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f7b027d45fd7484f6d0833823b98907e_FAQ

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

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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.lifetechnologies.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: 7277

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dcacff2565700c8f88f59cf4a16f9dfc_FAQ

Why is coupling efficiency important?

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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: 7278

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3332880692313818482a5a0286608ab6_FAQ

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: 7279

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f75dddd1e79826a219cb0bec217dc096_FAQ

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

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Please take a look at this list (https://www.lifetechnologies.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@lifetech.com to see if we can accommodate your request.

Answer Id: 7280

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b05b57f6add810d3b7490866d74c0053_FAQ

How do I calculate the melting temperature of my primers?

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

Answer Id: 7281

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701d804549a4a23d3cae801dac6c2c75_FAQ

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: 7282

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