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

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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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16f820033f5ce43855753b59efe50e9a_FAQ

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

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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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bc18bd5ef089709725ad04e9b39bd140_FAQ

Why did I get a lower yield of long PCR product with Platinum® Pfx than with a polymerase mix such as Elongase® enzyme?

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Answer

While enzyme mixes offer improved fidelity over Taq DNA Polymerase alone, Platinum® Pfx DNA Polymerase provides much higher fidelity since it is a proofreading polymerase exclusively. If yield is more important than fidelity, then an enzyme mix such as Platinum® Taq High Fidelity or Elongase® enzyme mix is a better choice of enzyme (since they contain a significant amount of Taq in addition to the proofreading polymerase). Platinum® Pfx does give high yields relative to other proofreaders, but may not give as great a yield as the enzyme mixes.

Answer Id: E3052

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9d316051100ef0c2c11dea0de1d0d8e2_FAQ

How should I adjust the Platinum® Pfx DNA Polymerase protocol if I am trying to generate an amplicon greater than 2 kb, or if I am starting with long PCR primers?

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Answer

To generate amplicons greater than 2 kb, use 2.5 units of Platinum® Pfx polymerase (instead of 1 unit), decrease the extension temperature to 68 degrees C, and increase the extension time to 1 kb/minute.

If long PCR primers are used with Platinum® Pfx polymerase, increase the magnesium concentration in the reaction to 1.5 mM.

Answer Id: E3053

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cd0709c9e25d9b088010e2e6124eb39c_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?

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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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5b5f8fa48225c7ffe5c7bfcd188cef48_FAQ

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

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Answer

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: E4290

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337cdf091765fed4a7afa0de58e0d347_FAQ

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

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Answer

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: E7266

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bd08d795d08f482150b59b7ec27d03d7_FAQ

What are the main steps in PCR?

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Answer

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: E7269

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3358bfd4156788bcf689aa8838c5d372_FAQ

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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df67b30462fa507a655f8176f130169d_FAQ

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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e0f629c17a9a4ea762ff4a4cac7ef39b_FAQ

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

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Answer

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: E7273

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bd40cefc09febe554f756e3a9ff6b6a2_FAQ

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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61f8074c86d7e6ecadf31adb5d12f90a_FAQ

Can you suggest some guidelines that will help me design my PCR primers?

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Answer

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: E7275

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c8e59776248f4b2335066a3cf97d3693_FAQ

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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65d594e2d7058b7c736fbd76360a9810_FAQ

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.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: E7277

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475e2054892183fe0cc93cb0338b7246_FAQ