Hotstar hifidelity pcr kit

Mammalian Gene Collection (MGC)-verified full length human KZNF cDNA clones were purchased from Thermo Fisher Scientific (Waltham, MA). PCR was performed to amplify ZNF cDNA using HotStar HiFidelity PCR kit (Qiagen, Valencia, CA). Primers were designed to amplify the region that includes KRAB and SCAN domains but not Zn finger motifs. Primers were also designed to include recognition sequences of the restriction enzymes as adaptors and three additional nucleotides to help binding of the restriction enzymes. The NCBI’s Primer-BLAST design tool was used to design primers and to calculate primer melting temperatures. After PCR, amplicons were digested with corresponding restriction enzymes to generate ligation-ready DNA fragments. For backbones, the pSG424 vector, which includes GAL4 DBD upstream of multiple cloning sites, was cut with the same restriction enzymes. Inserts were then ligated with the pSG424 backbone using Quick Ligation kit (New England BioLabs, Ipswich, MA). For transformation, 2µl of ligation reaction and One Shot MAX Efficiency DH5α-T1 competent cells (Invitrogen, Carlsbad, CA) were used according to the manufacturer’s protocol. Colonies were analyzed by restriction analysis and positive clones were sequenced (UW-Madison, Biotechnology Center) to verify correct incorporation and sequences of inserts.