PfxUltima with pENTR/SD/D-TOPO™ Cloning Kit with One Shot™ TOP10 Chemically Competent E. col - FAQs

View additional product information for PfxUltima with pENTR/SD/D-TOPO™ Cloning Kit with One Shot™ TOP10 Chemically Competent E. col - FAQs (12355020)

3 product FAQs found

Your Gateway-adapted TOPO vectors are supplied with a control template and control primers. Can I obtain the sequence of the control template?

The sequence of the control template is proprietary.

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

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.

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?

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