So yesterday’s experiment had some interesting results. Today I’m trying a different ligation approach. Instead of doing a 3-piece ligation, I’m doing two separate ligations: the unzipping DNA to the adapter, and then this product to the anchor. Here is the nomenclature for the reaction shown below:

• SpBR – pBR322 fragment digested with SapI restriction enzyme
• 5′-adapter – adapter DNA molecule with biotin attached to the 5′ end
• internal adapter – adapter DNA molecule with biotin attached to a dT within the molecule
• In keeping with my naming scheme, the products of these reactions will be labeled 5pBR and IpBR, for 5’adapter-pBR and internal-adapter-pBR respectively.