Heatshock Method!

  • 1x 50µL Vial of chemical competent E.coli strain of interest per transformation
    • One Shot™ ccdB Survival™ 2 T1R
    • Top10
    • DB3.1
    • XL10-gold
    • SHuffle T7
    • SHuffle T7 express LysY
    • SHuffle express
    • SHuffle T7 express
    • BL21
    • BL21(DE3)
    • BL21(DE3) pLysS
    • Rosetta2(DE3)
    • ArcticExpress(DE3)
    • C43(DE3)
  • 100ng of plasmid of interest per transformation

  • 1x LB-Agar plate with desired selection marker per transformation

  • S1 Benchtop Eppi Shaker
  • incubator closet
  • LB-Medium or SOC Medium

Day 1, date:

  • select program 6 on S1 Benchtop Eppi Shaker
  • get LB-Agar plate from cold room and leave on bench to get to roomtemp.
  • get desired Plasmid from -20°C and thaw on ice
  • get E.coli vial from -80°C and thaw on ice for 10min
  • add 100ng of plasmid to 1x 50µL vial of E.coli competent cells
  • mix by stirring gently with the pipette tip, do not vortex or mix by pipetting up and down
  • chill on ice for 10min
  • put vial into tabletop shaker and start program 6 (42°C, 45sec, no shaking)
  • select program 7 on S1 Benchtop Eppi Shaker
  • put vial back on ice until shaker has cooled to 37°C
  • add ___________µL LB or SOC Medium to vial (200 – 500µL)
  • mix by pipetting
  • put into shaker and start program 7 (37°C, 30min, 500 RPM)
  • spin down cells in vial
  • discard added LB Medium
  • resuspend cells in remaining 50µL
  • spread on prepared LB-Agar plate with desired selection marker with the help of a drigalski spatula or inoculating loop
  • turn upside down and incubate at ______°C for ______h (e.g. 37°C overnight, or 22°C over the weekend)

Day 2, date:

  • check LB-Agar plates for colonies