The mutants were constructed by PCR-based site-directed mutagenesis (Muta-directTM kit, Beijing SBS Genetech Co., Ltd., China).
Imperial College London
The wild CII263–272peptide (FKGE-QGPKGE), wild HA308–317peptide (YVKQNTLKLA), altered HA308–317peptide (YAKQATLALA), and irrelevant peptide (ALALTAQKAY) were synthesized by solid-phasetechniques (SBS Genetech). The purity was > 95%.
National University of Singapore
Ten peptides corresponding to the amino acids 20–39, 30–49, 42–61, 50–69, 60–79, 73–92, 88–108, 101–120, 111–130 and 124–144 of the Blo t 12.0101 protein were prepared by solid-phase synthesis with an automated peptide synthesizer, purified by high-performance liquid chromatography and (HPLC) and analysed by mass spectrometry(SBS Genetech, Beijing, China).
The PCR products were checked for effective amplification through electrophoresis in 1% agarose gels containing 1:20 GoldView (SBS Genetech Co., Beijing, China).
The RT-PCR products were resolved on 2% (w/v) agarose gel and stained by GoldView (Beijing SBS Genetech Co Ltd.) (fig. S1A).
University of Michigan
These oligonucleotides were designed to amplify a 250-bp segment of GAPDH that serves as an internal standard. The primers were purchased from SBS Genetech Co. Ltd, Beijing, China.
A Site‐Directed Mutagenesis Kit (SBS Genetech Co., Ltd) was then used to mutate the miR‐375 binding site in the 3′‐UTR of IGFBP3 and named as IGFBP3‐mutant‐type (MT) luciferase reporter plasmid.
Johns Hopkins University
Fluorescein isothiocyanate (FITC)‐labeled peptides (N‐terminal) usedthroughout this manuscript were synthesized from SBS Genetech(Beijing, China), and purified using reversed phase analytical high‐performance liquid chromatography (>99% purity).
The University of Hong Kong
The PCR reaction mixture consisted of 2 µL DNA (about 15 ng), 2.5 µL of 10 × PCR buffer, 1.5 µL of 25 mmol/L MgCl2, 1.5 µL of 2.5 mmol/L dNTPs, 1.5 U of Taq DNA polymerase (SBS Genetech Co., China), 2.0 µL each of 2.5 µmol/L IT1F and IT2R primers (synthesized by SBS Genetech Co., China) in a final volume of 25 µL.
University of California, Berkeley
Amplified products were run on a 2% agarose gel with GoldView (SBS Genetech Co., Ltd., Beijing) for visualization.
University of Toronto
Point mutations of RokC114-185 were generated using the site-directed mutagenesis kit (SBS Genetech).
Genomic DNA was extracted by Genome DNA Simax Kit (Beijing SBS Genetech Co., Ltd., China).
Site specific methylation within the gene promoter luciferase construct at the -148 site of upstream of transcript start site in Bdnf IV was generated using modified oligonucleotides (SBS, Genetech Co., Beijing, China). The modified oligonucleotide contained a −148 site specific methylated or an unmethylated CpG dinucleotide and spanned 15 bps on either side of the site. PCR was carried out according to the protocol in the Muta-directedTM mutagenesis kit (SBS, Genetech Co.).
Seoul National University
Each 25 µL reaction mixture contained 50 ng DNA, 5 pmol of each primer, 2 µL PCR buffer [100mM Tris (pH 8.3), 500 mM KCl, 15 mM MgCl2], 250 µM of each dNTPs and 0.5-1 unit Taq DNA polymerase (SBS Genetech Co, Beijing, China).
Total RNA was then extracted using Redzol reagent (SBS Genetech Inc., Beijing, China)
University of Wisconsin, Madison
Due to the high content of phenol and polysaccharides in pear leaves, genomic DNA was then purified using a purification kit from SBS Genetech Co. Ltd., China.
Shanghai Jiao Tong University
RNA was isolated using the Total RNA Isolation Kit (Beijing SBS Genetech Co., Ltd.) from mycelia of WT and its derivative ΔctcS mutant strains grown two days in fermentation medium.
University of Melbourne
After DNA extraction, extracts were loaded on 1% agarose gel in 0.5x Tris-Acetate-EDTA (TAE) buffer with GoodView Nucleic Acid Stain (SBS Genetech Co., Ltd., China) and were electrophoresed (100 V).
All primers were designed by Primer 5.0 software and synthesized by SBS Genetech (Beijing, China).
The University of New South Wales
We checked PCR ampli-cons with an electrophoresis in 0.7% agarose gelsstained using GoodView (SBS Genetech, Beijing, China)for visualization in UV light.
University of British Columbia
Gap26 and10panx1 were synthetized by SBS Genetech (Beijing, China).
University of Queensland
Reverse-transcription of total RNA into the first-strand cDNA was performed by an RT-PreMix kit (SBS Genetech) and a universal dT3AP [oligo(dT)-containing adaptor primer], which were directly used as templates for RACE (3 rapid amplification of cDNA ends) with primers MeuICK-F1 and 3AP (Figure 1A).
Desired mutations were introduced to the N-terminal double c-Myc–labeled human GLP-1R gene in pDONR201 (Invitrogen) via the Muta-directTM kit (Beijing SBS GenetechCo., Ltd., China)
University of California, San Diego
To make 5D, 5D-L, 5D-a, ASF-L and Tra2b-L series of constructs, sense and antisense oligos containing a full or truncated XhoI and EcoRI restriction sites were synthesized (SBS Genetech, Beijing) and diluted in the linker buffer (50 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM EDTA) at a final concentration of 100 nM.
University of Zurich
ISSR primers (SBS Genetech Co. Ltd, Beijing, China) were screened using the six samples of O. sinensis collected from the six spatially and clearly separated populations.
University of Malaya
All the oligonucleotide probes and target were procured from SBS Genetech Co., Ltd (Beijing, PRC)