Pfu ultra 2 hs dna polymerase

The wild-type (WT) human CaSR cDNA was cloned between the Kpn I and Xba I sites of pcDNA3.1 (+). All mutants were generated in pcDNA3.1(+)-WT CaSR. The Quikchange II site-directed mutagenesis protocol was used to introduce the point mutations T888A and T888M, as described previously (Lazarus et al., 2011 (link)): briefly, pairs of complementary or overlapping primers (30–40 bases) were designed to encode the required mutation with flanking wild-type sequences of ∼15–20 bases. The template DNA was amplified for 18 cycles with Pfu Ultra II HS DNA polymerase (Agilent Technologies, USA). Following digestion of parent DNA with Dpn I, amplified DNA was transformed into DH5α Escherichia coli cells.
The hCaSR/rmGluR1/rmGluR1 chimeric receptor construct was described previously (Mun et al., 2004 (link)). It encodes a class C GPCR protein that contains the hCaSR VFT domain (residues 1–540) appended to the rat mGluR1alpha Cysteine-rich domain, heptahelical domain, and intracellular C-terminus (residues 524–1199).
The identities of all mutants were confirmed by DNA sequencing (Australian Genome Research Facility, Sydney, NSW, Australia).

To generate lentiviral vectors expressing the NPRL2 mutants 34*, L105P, T110S, and D214H, QuickChange site-directed mutagenesis was used to mutate our pLVX-FLAG-NPRL2-IRES-zsGreen1 vector. PCR amplification was performed using PfuUltra II HS DNA polymerase (Agilent Technologies, Inc., Santa Clara, CA, USA) and oligonucleotide primers listed in Table S1. All plasmids were validated by sequencing performed on the SANGER Sequencing platform at the CHU de Québec-Université Laval Research Center.

Human DYK tagged O-GlcNAc transferase (OGT, OHu54262) and pig Pgc1α-myc (OSe00030D; p.C430S) clones were obtained from GenScript. Pgc1α was subcloned into pEGFP-N1 using BamHI and XhoI restriction enzymes. Site-directed mutagenesis was performed using Pfu Ultra II HS DNA polymerase (Agilent) and DpnI (Fermentas). The following primers were used to create p.430C variant in pig Pgc1α (PGC1aSer430Cys-F 5’-ccacagactcagaccagtgctacctgaccgagacgtcggag-3’ and PGC1aSer430Cys-R 5’-ctccgacgtctcggtcaggtagcactggtctgagtctgtgg-3’). The stop codon was eliminated to overexpress Pgc1α-myc-GFP using PGC1aEGFP-F 5-catctcagaagaggatctgttggatccaccggtcgccacc-3 and PGC1aEGFP-R 5-ggtggcgaccggtggatccaacagatcctcttctgagatg-3.