pSpark®

• Selecting the right pSpark®
• Low amount DNA cloning
• Cloning of long DNA fragment
• pSpark® and your time
• Sample request by email
• Beyond Pfu

The cloning efficiency varies significantly according to the size and sequence of the PCR product. The pSpark® DNA cloning systems are designed and tested for routine cloning of PCR products up to 9 kb in length with high efficiency.

When you have the challenging task of cloning long PCR fragments, it is specially important to remove impurities (e.g. primer dimers) or spurious bands from inefficient amplification. Thus, analysis of the PCR products on a gel prior to performing the ligation reaction, is a necessary step for cloning long PCR fragments. If gel analysis reveals inefficient production of the desired PCR product or reveals the presence of non specific products, it is strongly recommended to gel-purify the PCR product of interest and to quantify it in order to prepare an optimal cloning reaction. This reduces the number of white colonies containing inserts other than the desired PCR product.

Furthermore our R&D scientists have found that for large inserts (more than 7 kb) insert to vector ratios of 3:1 to 1:1 produce more colonies than insert to vector ratios from 5:1 to 3:1 that are optimal for inserts of 0.1 kb to 4 kb. This initial finding awaits confirmation for other large inserts, but for large inserts (> 7 kb) an insert to vector ratio of 3:1 represents a good starting point for cloning into pSpark® DNA cloning systems. If a gene of more than 9 kb is to be cloned we strongly recommend the use of the transcription-free pSpark® DNA cloning vector with low copy number to help insert stabilization.

The use of competent cells with a transformation efficiency of more than 5x107 cfu/µg is strongly recommended for cloning of long PCR products. pSpark® DNA cloning systems are compatible with any E. coli strain. Several E. coli strains have been developed to help stabilization of unstable DNA. For example, SURE® E. coli cells from Stratagene or Clean Genome® E. coli cells from Scarab genomics LLC are claimed to stabilize inverted repeated sequences and plasmid rearrangements during propagation. Although we have not tested them for cloning unstable DNA, they may be an alternative if cloning such problematic DNA. Also, XL10-Gold® from Stratagene has been designed for transformation with very large plasmids.