pSpark®

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

A highly relevant property of pSpark® DNA cloning system is that it is the only DNA cloning system for cloning less than 0,5 ng of insert. pSpark® cloning requires about 5-10 times less DNA than any other cloning kit available on the market. As a rule of thumb use 5 ng of insert per kilobase, that is 2.5, 5, 10, 15, 20 or 25 ng of insert for an optimal ligation of an insert of 500 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp or 5000 bp, respectively.

If initial experiments with your PCR product are suboptimal, ratio optimization may be necessary. Ratios of insert to vector from 5:1 to 1:1 provide good initial parameters. pSpark® DNA Cloning Systems have been optimized using an insert to vector ratio of 5:1. Optimal ratio of insert to vector is from 3:1 to 5:1. However, ratios of 0,2:1 to 50:1 have been used successfully.

Our R&D scientists have found that for large inserts (more than 7kb) 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 (>7kb) an insert to vector ratio of 3:1 represents a good starting point for cloning into pSpark® DNA cloning systems.See cloning of long DNA fragments.

The concentration of PCR product should be estimated either by comparison to quantitative DNA mass standards on an agarose gel, by absorbance at 260nm/280nm quantification using for example a Nanodrop™ Spectrofotometer or by using a fluorescent assay.

The pSpark® DNA cloning vectors I, II and IV have about 3,0 kb and are supplied at 4 ng/µL; the pSpark® DNA cloning vector IV has about 4,0 kb and is supplied at 5,3 ng/µL and the pSpark® DNA cloning vector V has about 3,4 kb and is supplied at 4,5 ng/µL. In all cases only 1 µL is used for a cloning reaction. To calculate the appropriate amount of PCR product (insert) to include in the ligation reaction, use the following equation:

Example of amount of insert calculation

How much 1 kb PCR product should be added to a ligation in which 4ng of pSpark® DNA vector (3.0 kb) will be used if a 5:1 insert to vector ratio is used?