Insert Priming Sites and Convert Single-Copy Clones to Multi-Copy-Inducible
The EZ-Tn5™ <oriV/KAN-2> Insertion Kit is designed to simplify and speed up both DNA purification and sequencing of existing single-copy BAC and fosmid clones by integrating CopyControl™ capability. Use a short, one-step in vitro reaction to randomly insert a single EZ-Tn5™ <oriV/KAN-2> Transposon containing an inducible, high-copy origin of replication (oriV) into the target DNA. Then, transform TransforMax™ EPI300™ or EPI300v-T1R Electrocompetent E. coli (sold separately) with an aliquot of the reaction and select for the kanamycin marker encoded by the EZ-Tn5™ Transposon.
One reaction will generate thousands of EZ-Tn5 <oriV/KAN-2> Transposon insertion clones. Each clone not only contains oriV and a kanamycin marker, but primer binding sites at the ends of the inserted transposon for sequencing. Amplify selected clones by adding the CopyControl™ Induction Solution and purify microgram quantities of template DNA from as little as 1ml of culture. Use the primers provided in the kit to sequence bidirectionally from the ends of the transposon and save the time and money usually spent subcloning or making custom primers.
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Figure 1. The process for generating EZ-Tn5™ <oriV/KAN-2> Transposon insertion clones for high yields of DNA and bidirectional sequencing. |
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