pENTR™/D-TOPO™ Cloning Kit, with One Shot™ TOP10 Chemically Competent E. coli

The sequence of the control template is proprietary.

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.

There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.

After generating your attB-PCR product, we recommend purifying it to remove PCR buffer, unincorporated dNTPs, attB primers, and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the Donor vector in the BP reaction and may increase background after transformation into E. coli, whereas leftover PCR buffer may inhibit the BP reaction. Standard PCR product purification protocols using phenol/chloroform extraction followed by ammonium acetate and ethanol or isopropanol precipitation are not recommended for purification of the attB-PCR product as these protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products. We recommend a PEG purification protocol (see page 17 of the Gateway Technology with Clonase II manual). If you use the above protocol and your attB-PCR product is still not suitably purified, you may further gel-purify the product. We recommend using the PureLink Quick Gel Extraction kit.

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.