Xl site directed mutagenesis kit

To generate luciferase reporters with varying lengths of the KRAS 3′ UTR, the construct, pGL4.75 KRAS#13 mLCS1, which was previously generated by Dr. Lena J. Chin [10 (link)], was used as a template. This template contains a 3910 bp region of the KRAS 3′ UTR originally cloned from DNA isolated from human genomic DNA. To generate the full-length KRAS 3′ UTR vector (pKRAS), we amplified the remaining 671-nt from the 3′ end of the 3′ UTR of KRAS separately from HeLa genomic DNA and then annealed it to the pGL4.75 KRAS#13 mLCS1 template using overlapping PCR with the Expand High Fidelity PCR System (Roche) and the primers listed below (Table 1). 3′ UTR truncation constructs were generated from the pKRAS vector using the primers listed below (Table 1). Each 3′ UTR fragment was amplified with Phusion High-Fidelity DNA polymerase (NEB), and cloned into the XhoI and NotI sites in the dual-luciferase vector, psiCHECK-2 (Promega). Deletions and mutations of the KRAS 3′ UTR fragment were created using PCR-mediated deletion as described in Hansson et al. [48 (link)], and site-directed mutagenesis using an XL Site-Directed Mutagenesis Kit (Agilent) and the primers listed below (Table 1). Each construct was confirmed by sequencing using the primers listed in Table 2.