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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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: E7273

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

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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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Is there anything to prevent AmpliTaq Gold™ DNA polymerase from extending from the 3’ end of a TaqMan™ probe in a 5’ nuclease assay?

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Yes. There is a phosphate group on the 3' end of all TaqMan™ probes that prevents such extension.

Answer Id: E1408

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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: E7274

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How are these oligos quality controlled?

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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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What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?

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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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Does Platinum™ Taq DNA Polymerase High Fidelity enzyme mix leave 3’ A-overhangs on the PCR product for subsequent cloning into a TOPO™ TA or original TA vector?

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Answer

Yes, the enzyme mix leaves 3’ A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3’ A-overhangs to the product for TA cloning.

Answer Id: E7268

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

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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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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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How does TA Cloning™ work?

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Answer

Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in our TA Cloning™ kits have single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

Answer Id: E4061

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What is the expected half life of AmpliTaq™ DNA Polymerase at 95 degrees C?

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Answer

The half-life of AmpliTaq™ DNA Polymerase at 95 degrees C is 40 min. During PCR, the sample is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95 degrees C is approximately 100 cycles.

Example: AmpliTaq™ DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.

Answer Id: E1139

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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.thermofisher.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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