pcDNA4c/His-Xpress-HA-hBIG1(E793K)
(Plasmid #79433)

• Purpose: Expresses N-terminally His6, Xpress and HA-tagged BIG1(E793K) in mammalian cells
• Depositing Lab: Kazuhisa Nakayama
• Publication: Shin et al Mol Biol Cell. 2004 Dec;15(12):5283-94. Epub 2004 Sep 22.

Backbone

• Vector backbone: pcDNA4c
• Backbone manufacturer: ThermoFisher Scientific
• Vector type: Mammalian Expression
• Selectable markers: Zeocin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Insert is somewhat toxic to E. coli. Requires long incubation time to see colonies after transformation and only small colonies should be chosen for use. Fresh colonies (direct from incubator; not stored at 4C) must be used for growth in liquid culture. See depositor’s comments for additional info
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: BIG1
• Species: H. sapiens (human)
• Mutation: changed Glutamic acid 793 to Lysine
• Entrez Gene: ARFGEF1 (a.k.a. ARFGEP1, BIG1, P200)
• Tags / Fusion Proteins:
  • HA (N terminal on insert)
  • His6 (N terminal on backbone)
  • Xpress (N terminal on backbone)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

It is tricky to amplify the plasmid. After transformation of the plasmid into E. coli, not only the transformants grow very slowly (usually, it takes more than 20 h for the transformants to form visible colonies on ampicillin‐containing plates and colony size is smaller than usual one), but also their viability is drastically decreased during storage at 4C
. Therefore, we routinely use fresh transformants for plasmid preparation. The plasmid yield decreases with the progress of the day after transformation. It is not recommended to make a pre-culture (~ 2ml) from a single colony for large scale culture. We strongly recommend that after transformation and recovery culture (for 1hr at 37C), bring all transformants to large scale culture (~ 200 ml) directly containing ampicillin.