Team:LMU-Munich/Notebook/Protocols/7 Measurement of competence



Measurement of competence

  • Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
    • This is at 10 pg/μl or 10-5 μg/μl
    • This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE
  • Hold on ice 0.5 hours
  • Heat shock 60 sec at 42°C
  • Add 250 μl SOC
  • Incubate at 37°C for 1 hour in 2 ml centrifuge tubes rotated
    • using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
    • For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
    • Ampicillin and kanamycin appear to do fine with 1 hour growth
  • Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
    • Good cells should yield around 100 - 400 colonies
    • Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
    • We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA