What is a suitable DNA template for TLAD?
The criteria defining a suitable dsDNA template consist of the following: • Size between 100-1000 bp. Anything smaller than 100 bp won’t be efficiently recovered by the Qiagen columns used in the TLAD method. Sizes between 1-5kb are probably permissible, though TLAD has not been tested extensively with that size range. Sizes >5kb should probably be amplified with an alternate method, such as strand displacement. • Absence of 3′ phosphate groups. Terminal transferase requires free 3′ hydroxyl (-OH) groups in order to add the homopolymer tail. Some DNA fragmentation methods (sonication, nuclease digestion, and yes, certain restriction digests) leave behind 3′ phosphate groups. Use the optional CIP method to remove these groups. • Low incidence of 3′ recessed ends. Terminal transferase tails best with dsDNA with either blunt or protruding 3′ ends. Sonication and certain restriction digests can generate dsDNA with a significant proportion of 3′ recessed ends. If yields are low, fill-in of
