Gene FAQ

  • What quality controls of gene products have been done?

    • - Agarose gel electrophoresis (structure)
    • - UV quantification (concentration)
    • - Use universal primers to amplify the sequences by PCR (size validation)
    • - Full-length sequence confirmation
    • - Plasmid stability tests
  • What if you can't obtain the clone from transformation into E. coli strains?

    • - Use a positive control plasmid
    • - Analyze DNA concentration using 1% agarose gel electrophoresis.
    • - Optimize transformation condition, increase the DNA and E. coli amount.
    • - Contact technical support
  • How to confirm ORF clone?

    • - Use universal primers to amplify the sequences by PCR and validate the size. Universal primers please refer to accompanying Certificate of Analysis (COA)
    • - Use universal primers to sequencing. Universal primers please refer to accompanying COA.
    • - Digest pCMV-vector with restriction enzyme for which is listed on the website or accompanying COA.
  • Why your actual sequencing results do not match the instruction of your website?

    • - About the cDNA nucleotide sequence, there is a detailed sequence description, please read the instructions carefully before buying.
    • - If a mistake by Sino Biological Inc. results in an order with mutation clone, Sino Biological Inc. will either ship a replacement at no charge or credit your account with the full purchase price.
  • Have you done expression test of each gene?

    • - Sino Biological Inc. is in progress for testing Flag-tagged expression-ready clones.
  • Why there are differences between your actual sequencing data and GeneBank sequences?

    • - Native mutations, polymorphisms, alternative splicing
    • - Artificially introduced changes by PCR cloning process.
  • What enzyme restriction sites were used to clone your ORF into the vector?

    • - Each ORF clone has different restriction enzymes sites.
    • - Please refer to the website or accompanying COA for detailed enzyme restriction sites of each product.
  • What's the resistance selection for cDNA clone?

    Vector Antibiotic In Bacterial Antibiotic In Mammalian Cell
    pMD18-T Ampicillin None
    pUC19/cPUC19 Ampicillin None
    pGEM-T Ampicillin None
    pCMV3-untagged Ampicillin Hygromycin
    pCMV3-C/N-Myc      pCMV3-C/N-HA
    pCMV3-C/N-FLAG      pCMV3-C/N-His
    Kanamycin Hygromycin
  • I cannot find the gene that I'm interested in in your website. Can you clone the gene for me? How much will it cost?

    • - Yes, please provide the GenBank accession, gene name, nucleotide or amino acid sequences
    • - It takes about 2~7 weeks to complete depending on the nucleotide sequence.
    • - No charge
  • Can I purchase the control vector?

  • Why no protein can be detected after gene expression?

    • 1) Compatible expression vector for host (eg. pCMV-vector for mammalian cell expression
    • 2) Need optimize the conditions of DNA-Cell transfection and culture condition.
    • 3) Protein subcellular location (secreted, cell membrane, cytoplasm or nucleus)
    • 4) Method of cell lysis
    • 5) Sensitivity of detection methods
  • What is Hygromycin concentration in mammalian cells? How to determine antibiotic sensitivity?

    • - Depending on the cell types, culture medium, growth condition and cell metabolism.
    • - Recommended concentration 50-500μg/mL.
    • - Test a range of concentrations for host cell line (refer to protocol below) :
    • 1) Cultured the untransfected host cells in the 96 well plates, about 100-500. Allow cells to adhere overnight.
    • 2) Substitute culture medium with medium containing varying concentrations of hygromycin (0, 50, 100, 200, 400 , 500μg/mL).
    • 3) Replenish the selective media every 3–4 days, and observe the percentage of survival cells.
    • 4) Count the number of viable cells at regular intervals to determine the appropriate concentration of hygromycin that prevents growth within 2–3 weeks after addition of hygromycin.
    Cell Specie Tissue Medium Hygromycin B(μg /ml)
    Hela Human uterus DMEM 200
    293 Human kidney DMEM 100
    B16 mouse melanoma RPMI 50-100
    C6 hat glioma DMEM 100-200
    PC1.0 hamster adenocarcinoma RPMI 100-200

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