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

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Answer

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

Answer Id: E6550

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a8d44f44c0dc6eb8cef79d01f45e20b1_FAQ

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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Answer

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

Answer Id: E6815

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527098967a126a7809f5773aa3c66c49_FAQ

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

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Answer

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: E6651

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c282cdd6154484d04bb8b2ce3e2275db_FAQ

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

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Answer

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: E6738

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c1164b31f0912756d36aac7f43b4c2b4_FAQ

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

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Answer

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: E6743

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2b7a1804c053424ca3c174ecc182a20f_FAQ

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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Answer

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: E6757

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651215f8f14034c194beb3d3e87861e3_FAQ

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

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Answer

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: E6762

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1965f6002ee7907d1285b8b43cd00231_FAQ

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

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Answer

You may have to try different ratios ranging from 1:1 to 15:1 insert:vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

Answer Id: E7645

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7f0ffc8664d35f9b979c64a966bd3453_FAQ

What is the best molar ratio of PCR product:vector to use for TOPO® TA cloning? Is there an equation to calculate the quantity to use?

Product FAQ

Answer

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO® cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

Answer Id: E7648

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42f13cc90bca8db8f0cdcf99d3966301_FAQ

I am using a Directional TOPO® Cloning vector for cloning my PCR product. I have screened a bunch of colonies but they all contain my insert cloned in the reverse direction. How can I solve this problem?

Product FAQ

Answer

Here are possible causes and suggestions:

- Incorrect PCR primer design: Make sure that the forward PCR primer contains the sequence, CACC, at the 5' end. The 4 nucleotides, CACC, base pair with the overhang sequence, GTGG, in the Directional TOPO® vector.
- Reverse PCR primer is complementary to the GTGG overhang at the 5' end: Make sure that the reverse PCR primer does not contain the sequence, CACC, at the 5' end.
- Use a thermostable, proofreading polymerase such as Accuprime™ Pfx DNA Polymerase (Cat. No. 12344024) to produce blunt-end PCR products.

Answer Id: E7649

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8efdf9a2b0fc425b9803e983e8f94bcc_FAQ