Library efficiency dh5α competent cells

The cA3 in the pCR-TOPO-II vector was restricted with BamHI (New England BioLabs) and EcoRI (New England BioLabs) restriction endonucleases. PCR primers were designed to introduce HindIII recognition site at the 5′ end of the gene and AgeI recognition site at the 3′end of the gene which eliminated the stop site. The PCR primers are 5′- CGGAAGCTTGCAGAGATGGCCAAGATTAACACCCAAGTCTCC-3′ and 5′-GGCGACCGGTGACTGCTCTTTGATCTCTGTTTTTGCGGC-3′. The pEYFP-N1 vector (Clonetech) was prepared for ligation by digesting with HindIII and AgeI. The PCR gene product was ligated into the pEYFP and the product was used to transform Library Efficiency® DH5α Competent Cells (Invitrogen). DNA sequencing confirmed the full length gene product in the pEYFP-N1vector. The hB3 gene, a gift from Bernd Wissinger, was cloned into pCDNA 3.1 (Invitrogen) for eukaryotic expression. Attempts to clone this gene into pEYFP-C1 or pEYFP-N1 to examine the cellular localization of the fluorescent tag resulted in the failure of heteromeric channel expression when co-expressed with untagged cA3. For this reason, all heteromeric channel studies were comprised of cA3 in pEYFP co-expressed with human hB3 in pcDNA3.1. Mutations were generated using primers designed with point mutations; mutagenesis was accomplished using QuikChange® (Stratagene, San Diego, CA). Full-length sequences were obtained for all mutant cDNAs.

The resultant PCR product was cloned into the pDONR/
Zeo gateway entry vector using the BP clonase according to
the supplier�fs directions (Invitrogen, USA). The recombinant
pENTER/Activin A entry clone was transferred into Library
Efficiency® DH5α™ Competent Cells (Invitrogen, USA)
by the heat shock method as described by the manufacturer.
Clones were cultured in Luria-Bertani (LB) broth overnight
and plasmid extraction was performed using the AccuPrepR
Plasmid Mini Extraction Kit (Bioneer, Korea). Recombinant
vectors were examined by PCR using the M13-F and Activin
-R primers which generated an amplicon of about 650 bp.
DNA sequencing of the inserted segment was done using
M13-F: 5'-GTA AAA CGA CGG CCA GT-3' and
R: 5'-AGC GGA TAA CAA TTT CAC ACA GGA-3' primers.

For PCR amplification of the genes of interest, DNA isolated with ChargeSwitch gDNA Mini Bacteria Kit (Invitrogen, Waltham, MA, USA) was used as template. Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) was used for amplification according to the manufacturer’s instructions; the primers are listed in Table S2. The gel-purified PCR products (GeneJET Gel Extraction Kit, Thermo Scientific, Waltham, MA, USA) and plasmid pSCrhaB2 [27 (link)] were double-digested with NdeI and HindIII (New England Biolabs). After ligation (Quick Ligation Kit, New England Biolabs), the mixtures were transformed into Library Efficiency DH5α™ Competent Cells (Invitrogen) and transformants were selected on LB plates supplemented with trimethoprim (50 µg mL⁻¹). Plasmids were extracted using ChargeSwitch-Pro Plasmid Miniprep Kit (Invitrogen) and the correct insert size was confirmed following NdeI/HindIII double-digestion by gel electrophoresis. Plasmids were introduced into B. cenocepacia J2315 by triparental mating as described [27 (link)]; transconjugants were selected on LB plates supplemented with trimethoprim (200 µg mL⁻¹).

The pDest17/TAT-SOX2 and pDest17/TAT-EGFP constructed by Gateway Technology (Invitrogen, Carlsbad, CA, USA). Expression clones were transferred into Library Efficiency DH5α Competent Cells (Invitrogen, Carlsbad, CA, USA) by the heat shock method as described by the manufacturer for confirming recombination and making glycerol stock for further uses. Confirmed clones were used for recombinant protein production.
Recombinant protein production was performed as previously described [30 (link)]. Briefly, pDest17/TAT-SOX2 and TAT-EGFP expression vector were transformed into E. coli strain BL21 competent cells (DE3; Novagen,WI, US). The transformed cells were cultured to reach an OD 600 ~ 0.8 and then induced by 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG, Fermentas, Lithuania). His6 was also used for protein purification. The His6-TAT-SOX2 and His6-TAT-EGFP proteins were purified by the Ni-NTA Fast Start Kit (Qiagen, USA) in denature condition. Immobilized SOX2 proteins eluted with 8 M urea (pH 3.5), then desalted by Tris (5 mM) that contained 50% glycerol and maintained at -20°C until use. The purified proteins were analyzed by SDS-PAGE, Coomassie blue staining (S1 Fig).

To clone additional MHC genes, we designed primers from grey mouse lemur (Microcebus murinus), thick-tailed bushbaby (Otolemur garnetti), and Philippine tarsier (Tarsius syrichta) Genbank sequences (Additional file 1: Table S1). Our PCR had an initial denaturation of 45 s at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 54 °C, and 1 min at 68 °C, and a final extension of 7 min at 68 °C. Using pGEM-T® Easy Vector (Promega, Madison, WI) and Library Efficiency® DH5α Competent Cells (Invitrogen, Life Technologies, Grand Island, NY), we cloned the PCR products following manufacturers’ instructions. In other primate species, these genes have much reduced diversity compared to MHC-DRB [16 (link), 80 (link)]. We therefore sequenced only 10–30 positive clones per gene, per individual, on ABI 3730xL Analyzer using Big Dye chemistry (Applied Biosystems®, Life Technologies, Grand Island, NY). We considered as alleles only sequences found in minimally three clones per PCR. We used MEGA 5.2 [83 (link)] to align and analyze sequences from MHC-DOA, MHC-DOB, MHC-DPA, MHC-DQA, and MHC-DRA.