Competent HT115

Competent HT115 kdorfman Mon, 12/20/2021 - 15:59
to make: aliquots
at this volume: uL
Innoculate mL LB tet
with mL overnight culture
Grow to OD595=0.4
Spin 3000rpm for 10 min at 4C
Resuspend in: mL cold 50mM CaCl2 GENTLY
Spin as before, 3000rpm for 10 min at 4C
Resuspend in: mL cold 50mM CaCl2 GENTLY
Use immediately or freeze by adding: mL sterile glycerol

  • Inoculate overnight culture in LB + antibiotic (TET for HT115(DE3) strain) (2-5ml). Shake overnight at 37C.

  • Inoculate 25 ml LB + antibiotic with overnight culture, 1:100 dilution. Grow cells to OD595= 0.4. Can grow cells in 50 ml sterile centrifuge tube.

  • Spin cells 10 min 3000 rpm at 4C.

  • Resuspend pellet in 0.5X original volume cold, sterile 50 mM CaCl2 (12.5ml). Resuspend by GENTLY pipetting up and down a few times with a wide bore pipet--no vortexing.

  • Incubate on ice 30 min.

  • Spin as before at 4C.

  • Resuspend pellet as before in 0.1X original volume CaCl2 (2.5ml). Keep cells cold (4C).

  • Use 50-200 ul for transformation. Cells can be used as is for up to three days (stored at 4C).

  • The cells can be frozen by: adding glycerol to final concentration of 10%, rapid freezing on dry ice/EtOH, and storing at –80.