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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What does “SD” stand for in pENTR/SD/D-TOPO® vectors?

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SD stands for Shine Dalgarno, which is a bacterial ribosomal binding site for efficient initiation of expression in E.coli.

Answer Id: 6550

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I am cloning my gene of interest into the pENTR/SD/D-TOPO vector, but plan on expressing my gene in both E.coli and mammalian cells. Will the Shine-Dalgarno sequence interfere with mammalian expression?

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No, the Shine-Dalgarno sequence does not adversely affect mammalian expression when used in an appropriate mammalian DEST vector.

Answer Id: 6815

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Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?

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A. TA Cloning:
- This cloning method was designed for pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 of 15:1 ratio of Taq to proofreader polymerase will still generate enough 3’ A overhangs for TA cloning
- The recommended Life Technologies® polymerases include Platinum® Taq, AccuPrime™ Taq, Platinum® or AccuPrime™ Taq High Fidelity, AmpliTaq®, AmpliTaq Gold®, or AmpliTaq Gold® 360.

B. Blunt cloning:
- Use proofreading enzymes such as Pfx50, Platinum® or AccuPrime™ Pfx.

C. Directional TOPO cloning:
- Pfx50 or AccuPrime Pfx work well.

Answer Id: 6651

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What are the requirements for primer design when using directional TOPO® cloning?

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Please consider the following when designing your primers:

- The 3’ pcr primer cannot contain homology to the 5’ flap sequence GTGG.
- The enzyme you use must create a blunt-ended PCR product for cloning.
- Primers cannot contain 5’ phosphates, which will block the 5’ OH nucleophile reactive group.
- The reading frame must be considered when you are designing your primers.

Answer Id: 6738

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Can I use a Taq polymerase to generate my gene of interest for directional TOPO® cloning?

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No, your gene of interest must be amplified with a proofreading polymerase such as AccuPrime™ Pfx or Platinum® Pfx that leaves blunt ends for directional TOPO® cloning.

Answer Id: 6743

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I am searching for a cloning vector that has gateway compatible att sites present in the vector for later transfer to an expression vector. A directional cloning bias would be a huge advantage. Do you have any suggestions?

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We would suggest using one of our pENTR D-TOPO® kits for easy TOPO® cloning of your insert with the advantage of directionality.

Answer Id: 6757

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I’m getting low cloning efficiency with my directional TOPO® cloning reaction. What should I do?

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Here are suggestions to try to increase your cloning efficiency with dTOPO® cloning:

- Ensure that the 5’ primer has CACC and the 3’ primer does not have sequence similarity to GTGG.
- The molar ratio of PCR product: TOPO® vector used is critical to success.

We recommend using a 1:1 to 2:1 molar ratio, starting with a 1:1 of PCR product: TOPO® vector. The TOPO® cloning efficiency decreases significantly if the ratio of PCR product: TOPO® vector is <0.1:1 or >5:1. These results are generally obtained if too little PCR product is used (i.e., PCR product is too dilute) or if too much PCR product is used in the TOPO® cloning reaction. If the yield of the PCR product has been quantitated, the concentration of the PCR product may need to be adjusted before proceeding to TOPO® cloning. For pENTR™ TOPO® vectors, using 1-5 ng of a 1 kb PCR product or 5-10 ng of a 2 kb PCR product in a TOPO® cloning reaction generally results in a suitable number of colonies.

Answer Id: 6762

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