Linear DNA library construction
Linear DNA libraries were constructed by annealing two single stranded
DNA (ssDNA) oligos and extension by Klenow polymerase. Oligos were
ordered through FASMAC Inc., (Supplementary Table 1). A final
concentration of 3 μM of Display-F and Display-FLAG-random-R oligos were
used for the synthesis of FLAG-random library, as well as Display-F and
Display-10aa-random-R oligos for the synthesis of 10aa-random library
and Display-F and FLAG-control-R oligo for the synthesis of fixed FLAG
(DYKDDDDK) sequence for western blot analysis. Oligo pairs were mixed
with final 200 μM dNTPs in Klenow buffer (Takara Bio) and annealed by
heating at 92 ℃ for 30 seconds followed by cooling to room temperature.
Then 10U of Klenow Fragment (Large Fragment E. coli DNA
Polymerase I;l Takara Bio) was added to the reaction mix and an
extension step was performed at 37 ℃ for 1 hour, followed by
inactivation of the enzyme at 50 ℃ for 15 min. The Linear DNA libraries
(FLAG-random and 10aa-random) were further column purified using the
Wizard SV Gel and PCR Clean-Up System (Promega) and quantified by
Nanodrop2000c (Thermo Fisher Scientific).