How does AmpliTaq Gold™ DNA Polymerase differ from AmpliTaq™ DNA Polymerase?

Product FAQ

Answer

AmpliTaq Gold™ DNA Polymerase is a modified form of AmpliTaq™ DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold™ DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp™ 10X PCR Buffer I and/or GeneAmp™ 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp™ 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).

Answer Id: E1339

Was this answer helpful?

Yes
No
Thank you for your response

I’m getting low yield of my oligo upon reconstitution. What happened?

Product FAQ

Answer

The oligo may not have been fully solubilized. After addition of TE buffer, make sure the oligo was vortexed for a full 30 seconds and/or pipette up and down more than 10 times. Primers may be present along the sides of the tubs, so when resuspending the oligo, the sides of the tubes should be “rinsed” too.

Answer Id: E7294

Was this answer helpful?

Yes
No
Thank you for your response

I ordered a primer with restriction enzyme sites flanking the 3’ and 5’ ends of my oligo with desalted purification. When trying to subclone the PCR product, I get very few colonies. I have tested all conditions, and it seems to be the oligo causing the problem. Can you explain why this happened?

Product FAQ

Answer

Better purification of the oligos is recommended to provide you with full-length oligo sequence. Adding restriction sites adds on 10 or more bases to the basic 20-25-mer, making primers longer than 30 bases with a relatively low percentage of full-length sequences after desalting. Additionally, failure sequences occur at the 5’ end of the sequence as oligos are generated from 3’ to 5’ end. Therefore, restriction sites introduced at the 5’ end of primers can be compromised, resulting in missing bases.

Answer Id: E7295

Was this answer helpful?

Yes
No
Thank you for your response

I’m missing a nucleotide in the middle of my sequence. How could this happen?

Product FAQ

Answer

There are two possibilities that could occur in any round of extension when creating your primer:

1.The added base is not detritylated correctly, missing one base addition but allowing possible extension in the next round.
2.The trityl group was removed, but not coupled or capped correctly before addition of the next base, allowing the chain to continue.

Answer Id: E7296

Was this answer helpful?

Yes
No
Thank you for your response

How does TA Cloning™ work?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

When amplifying long PCR targets, is the concentration of the deoxynucleoside triphosphates (dNTPs) limiting?

Product FAQ

Answer

The concentration of dNTPs in a standard PCR amplification is 200 μM each, for a total of 800 μM. This total dNTP amount corresponds to 39 μg of dNTPs. This is a huge excess and, when generating long PCR fragments, is not a limiting factor during the PCR amplification, as the amount of target DNA generated is generally no more than 1 μg. More importantly, the reaction condition variables need to be monitored more closely in order for successful long PCR amplification to occur.

Answer Id: E1084

Was this answer helpful?

Yes
No
Thank you for your response

What does hot start PCR mean?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

How do I calculate the melting temperature of my primers?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

Why is coupling efficiency important?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

Can you compare the DNA polymerases you offer by fidelity, maximum amplicon length, and 3’ A-overhang?

Product FAQ

Answer

Please see the comparison below on the following criteria:
Enzyme, Relative Fidelity, Amplicon Length, and 3’ Overhang (+/-)
Taq, 1, <5 kb, +
Platinum™ Taq, 1, <10 kb, +
AccuPrime™ Taq, 2, <5 kb, +
Platinum™ Pfx, 26, <12 kb, -
AccuPrime™ Pfx, 26, <12 kb, -
Pfx50™, 50, <4 kb, -
Platinum™ Taq HiFi, 6, <20 kb, +/-
AccuPrime™ Taq HiFi, 9 <20 kb, +/-
AmpliTaq™, 1, <5 kb, +
AmpliTaq Gold™, 1, <5 kb, +
AmpliTaq Gold™ 360, 1, <5 kb, +

Taq error rate: 1 x 10-4 to 2 x 10-5 base/duplication

Answer Id: E7263

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response