Lab Manual

Protocol

Handling of genetic materials

• Stab culture
  • Inoculate the bacteria on an agar plate containing appropriate selective antibiotics and/or nutritional supplements within a few days after you received the plastic vial of stab culture.
  • Pick up bacteria from the middle part of stab culture but not those from the surface of the stab which are not really healthy.
  • CAUTION: The vial should be placed in a refrigerator (4°C) but never in a freezer, otherwise spoiled.
    
• BAC Related Information
• Microbe Genomic DNA Preparation
• Lentiviral vectors [link]

Sequencing & PCR primer

• Sequencing & PCR primers
• Primers for PCR of rRNA gene (Microbe Genomic DNA Information)
• List of STS Markers for Detection of Virus Genes

Protocol by using deposited matelials

• Expression and Purification of Recombinant Galectins
  

  Resource page	Protocol
  Lectin (GSB0048) – Gene Set Collection	Nishi, H. A note on expression and purification of recombinant galectins. Glycoforum Vol.23 (5), A15 (2020). DOI: https://doi.org/10.32285/glycoforum.23A15.

• Purification of Recombinant Tubulin by Dr. Etsuko Muto [pdf]
  

  Catalog #	Resource name	Description
  RDB12753	pFBD-Hs alpha1CGH/beta3CGF	Plasmid clone for baculoviral expression of alpha1 and beta3 tubulin.
  RDB12754	pFBD-Hs alpha1(K40R)CGH/beta3CGF	Plasmid clone for baculoviral expression of alpha1 and beta3 tubulin.
  RDB14530	pFBD-Hs alpha1 CFXaGH/beta3 CFXaGF	Expression vector of His tagged human tubulin alpha-1and Flag tagged human tubulin beta-3.
  RDB14531	pFBD-Hs alpha1(K40R) CFXaGH/beta3 CFXaGF	Expression vector of His tagged human tubulin alpha-1(mutant, K40R, constitutive active form) and Flag tagged human tubulin beta-3.

• Expression of heterologous proteins in Drosophila Schneider S2 (S2)
  

  Resource page	Protocol
  Empty Backbone for Insect Cell Experiment	A single plasmid transfection that offers a significant advantage associated with puromycin selection in Drosophila Schneider S2 cells expressing heterologous proteins. Iwaki, T., Castellino, F.J. Cytotechnology 57 (1), 45-49, (2008). PubMed PMID 19003171.

• Fluorescence in situ hybridization using bacterial artificial chromosome (BAC) clones
  

  Resource page	Protocol
  Chinese hamster genomic BAC library clone	Fluorescence in situ hybridization using bacterial artificial chromosome (BAC) clones for the analysis of chromosome rearrangement in Chinese hamster ovary cells. Cao, Y., Kimura, S., Itoi, T., Honda, K., Ohtake, H., Omasa, T. Methods 56 (3): 418-423 (2012). PubMed PMID 22100493.

• Visualization of sphingolipids
  

  Resource page	Protocol
  Genes involved in the sphingolipid signaling pathway	Dynamics of sphingomyelin-and cholesterol-enriched lipid domains during cytokinesis. Abe, M., Kobayashi, T. Methods Cell Biol. 137: 15-24 (2017). PubMed PMID 28065303.

• Optogenetic technology using cultured cell lines
  

  Resource page	Protocol
  Genetic Resources for Optgenetics Research	Myogenic Maturation by Optical-Training in Cultured Skeletal Muscle Cells. Asano, T., Ishizuka, T., Yawo, H. Methods Mol. Biol. 1668: 135-145 (2017). PubMed PMID 28842907.

• Cell-free protein synthesis with noncanonical amino acids using Escherichia coli cell extract
  

  Resource page	Protocol
  E. coli strain for producing recombinant proteins incorporating synthetic amino acids	Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains. Seki, E., Yanagisawa, T., Yokoyama, S. Methods Mol. Biol. 1728: 49-65 (2018). PubMed PMID 29404990.

• Cell cycle observation by using Fluorescence Ubiquitination Cell Cycle Indicator (Fucci)
  

  Resource page	Protocol
  Fucci – Fluorescent Ubiquitination-based Cell Cycle Indicator	Methods to study the proliferation and differentiation of cardiac side population (CSP) cells. Sereti, K.I., Oikonomopoulos, A., Unno, K., Liao, R. Methods Mol. Biol. 1036: 95-106 (2013). PubMed PMID 23807790.

• Transduction of gene to fission yeast by pDUAL and pDUAL2 vectors
  

  Resource page	Protocol
  Expression Vector Backbone of S. pombe	Heterologous gene expression by chromosomal integration in fission yeast. Matsuyama, A., Yoshida, M. Methods Mol. Biol. 824: 433-450 (2012). PubMed PMID 22160913.

• E. coli-based in-cell NMR experiment
  

  Resource page	Protocol
  Thermus thermophilus Expression plasmid	In-cell NMR of intrinsically disordered proteins in prokaryotic cells. Ito, Y., Mikawa, T., Smith, B.O. Methods Mol. Biol., 895: 19-31 (2012). PubMed PMID 22760309.

Reference of Genetic Materials

• List of references of genetic materials (promoters, plasmid vectors and so on).
• List of RDB Clones in Publications
  RDB Clones in Publications is a list of scientific journal and patent application in which provided resources of RIKEN BRC DNA Bank were used. When you publish your research results in a scientific journal, please refer to the origin of materials that were provided from RIKEN BRC and used in your research subject. We appreciate your cooperation.

(MAN0000e 2011.01.12 T.M.)

2023.06.08