Large inserts should be gel-purified prior to cloning. Gel purification of long PCR products often improves efficiency, as smaller non-specific bands that may not be visible on the gel will interfere with cloning of the insert of interest. However, the TOPO XL-2 Complete PCR Cloning kit is supplied with the PureLink Quick Gel Extraction and PCR Purification Combo Kit for added flexibility allowing for faster PCR purification when inserts are smaller, while primers and dNTPs still need to be removed.
Keep in mind that the TOPO reaction does have a strong size bias, and that any smaller fragments present in the PCR reaction would clone much more efficiently than the fragment of interest, even if the smaller fragment is not readily visible on a gel. Therefore, it is highly recommended that the PCR product of interest be gel-purified.
Answer Id: E16001
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