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

Product FAQ

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 Thermo Fisher Scientific 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

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

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

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

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

What are Value Oligos?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

I’m seeing high molecular weight EtBr stainable material left in wells. Why is this happening?

Product FAQ

Answer

This artifact occurs when either too many cycles were performed or too much DNA is added to the reaction. Try heating to 65 degrees C and putting sample on ice before loading.

Answer Id: E7292

Was this answer helpful?

Yes
No
Thank you for your response

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?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

How do I determine the percentage of full-length oligonucleotide?

Product FAQ

Answer

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: E7279

Was this answer helpful?

Yes
No
Thank you for your response

Do you have any resources to help design primers?

Product FAQ

Answer

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

Answer Id: E7276

Was this answer helpful?

Yes
No
Thank you for your response