Pfx dna polymerase

Total RNA was isolated from LNCaP, DU-145, and DLD-1 cells using RNeasy Minikits (Qiagen). cDNAs were synthesized using AMV reverse transcriptase (Promega) and CXADR isoforms of PCR products were amplified using the following primers: general sense primer, 5′-ATGGCGCTCCTGCTGTGCTTCGTG-3′; isoform 1 revers primer, 5′-CTATACTATAGACCCATCCTTGC-3′; isoform 2 and 3 reverse primer, 5′-CTACCTTAGCAGGTGGGAGAGTC-3′; isoform 4 reverse primer, 5′-TTACTGCCGATGTAGCTTCTGGC-3′; isoform 5 reverse primer, 5′-TTATACAACTGTAATTCCATCAG-3′. Full-length human CXADR cDNA fragments of isoform 1 and 5 containing NheI and XhoI sites were generated by PCR using DU-145 cDNA, PfxDNA polymerase (Invitrogen) and the following primers: general sense primer, 5′-TATGCTAGCCACCATGGCGCTCCTG-3′; isoform 1 reverse primer, 5′-TATCTCGAGTACTATAGACCCATCCTTGCT-3′; isoform 5 reverse primer, 5′-TATCTCGAGTACAACTGTAATTCCATC-3′. The NheI/XhoI fragments of CXADR isoforms were inserted into an NBEX-Flag vector. The NBEX-Flag vector was constructed by insertion of the C-terminal Flag-tag sequence into pcDNA3.1/Hygro(+) cleaved with NheI and PmeI. The sequence of all expression vectors was confirmed by sequencing. MKN-7 cells were transfected with the expression vectors using the Lipofectamine reagent and selected with hygromycin.

Plasmid DNA templates for full-length Etv1, Etv4, Etv5, and Fev were obtained from the Mammalian Gene Collection (I.M.A.G.E. Consortium Clone IDs 30345383, 3854349, 3050350, and 4130242, respectively) (47 (link)). Regions corresponding to the core Ets domains were amplified by PCR using Pfx DNA polymerase (Invitrogen, Paisley, UK). PCR products were ligation independent cloning into the pNIC28-Bsa4 expression vector (GenBank^TM accession number EF198106, encompassing a tobacco etch virus (TEV)-cleavable (shown by *) N-terminal His₆ tag MHHHHHHSSGVDLGTENLYFQ*SM), as described elsewhere (48 (link)). The initial Etv1 construct contained two primer-incorporated mutations (Y329S and P427S) and thus were designated Etv1^Y329S-P427S. A subsequent construct containing the wild-type sequence is referred to as Etv1. Additional mutants were generated in a full-length Etv1 construct using the megaprimer method (49 (link)). The Gly-326–Asn-429 fragments of the Etv1 mutant derivatives were then subcloned for bacterial expression into pNIC28-Bsa4 as described and confirmed by sequencing. Expression plasmids were transformed into BL21 (DE3) Rosetta-R3 (48 (link)) or, when indicated, into Rosetta-gami^TM 2 (Novagen®).

Primers (MWG) to clone MtSerB2 and its variants contain BamHI and HindIII restriction sites in the forward and reverse primers respectively. PCR reactions were carried out using Pfx DNA polymerase (Invitrogen). The product corresponding to the native enzyme was cloned into pET23a vector (Novagen) and called pET23a:MtSerB2. Recombinant Hexa-his-tagged MtSerB2 was expressed using E. coli C41 (DE3). The pET23a:SerB2_ACTD (residues 1–165) and pET23a:SerB2_PSPD (residues 165–409) constructs corresponding to the ACT and PSP domain mutants were used to over-express the mutants in E. coli BL21 (DE3) and C41 strains respectively. Other mutants that were generated are G18A, G108A, D185N, D187N, S273A, K318A, D341N, D345N, D185N/D187N & D341N/D347N. Mutants were over-expressed similar to the wild-type except that D187N and S273A were grown at lower temperature i.e., 25°C instead of 37°C to overcome problems of solubility of protein. Integrity of all constructs was verified by sequencing.