Technicians place the cells on ice for up to 10 minutes. Then, they add four microliters of DNA into the cell solution and mix well using a pipette tip. They place the solution back on ice for 30 minutes, heat shock the cells using a 107.6 degrees Fahrenheit water bath for 45 seconds, and then put back on ice for an additional two minutes. Finally, technicians spread the cell solution onto Kan LB medium plates, and incubate at 98.6 degrees Fahrenheit for 6 hours. Protein expression is induced using isopropyl-beta-D-1-thiogalactopyranoside.
Technicians prepare a cell culture medium without serum and assess viability prior to performing electroporation, using trypan blue stain followed by microscopic observation. Samples are placed in an apparatus called an electroporator. According to Martin J. Tymms in "Transcription Factor Protocols," as voltage increases, cell viability decreases. The electroporation conditions that reduce viability by 50 percent are also the conditions at which gene expression is near maximal. "Gene expression" refers to the production of specific proteins, according to relating DNA segments.
This protocol involves the transformation of E. coli strain BL21, grown in Luria broth medium at 98.6 degrees Fahrenheit. Citrate carrier (CitS) were expressed in E. coli BL21 adding the chemical carbenicillin at concentration of 100 mg per ml. Citrate transport activity in the recombinant strains was detected as blue halos around colonies on Simmons citrate agar plates. Alkaline phosphatase activity was detected as blue colonies on Luria broth agar plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (toluidine salt; XP) at a concentration of 40 mg per ml.