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Whitepaper: Single-tube DNA purification and cloning using ultrafiltration devices

Posted: 24 May 2016 | | No comments yet

Traditional methods for subcloning DNA fragments or shuttling sequences between vectors involve the ligation of restriction digest fragments…

Traditional methods for subcloning DNA fragments or shuttling sequences between vectors involve the ligation of restriction digest fragments from chromosomes, plasmids, cosmids, or PCR products into specific sites on a selected cloning vector.

In order to desalt, remove primers or adapters, or concentrate DNA fragments, researchers generally use gel electrophoresis and precipitation steps that are labor-intensive and time-consuming. In an effort to develop a more streamlined process, we compared traditional methods of subcloning PCR-generated fragments against a rapid procedure using Nanosep® centrifugal ultrafiltration devices. After optimizing the parameters for effective target DNA purification, we synthesized PCR products with terminal adapter sequences containing restriction sites. These adapters were digested, generating the appropriate cohesive ends for ligation. The resulting DNA products were concentrated and desalted while simultaneously allowing the primers and adapter fragments to pass into the discarded filtrate during a five-minute centrifugation step. The resulting ligation-ready DNA fragments were ligated to digested vector directly within the ultrafiltration device, and then transformed into competent cells. Using Pall Life Sciences Nanosep devices, we were able to achieve significant savings in time, labor, and starting materials, while the resulting yield of transformed recombinants roughly equaled that of the traditional techniques.

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