User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/09/09

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Competent cells: TOP10 & DB3.1

  • 10 mL ON culture is used to inoculate 2·10 mL of LB, 50 μL per 10 mL.
  • Cultures are grown @ 37 °C untill an OD600 of 0.2 ~ 0.3 is reached.
  • Cultures are spinned down 5' @ 4000 rpm, 4 °C
  • Supernatant is removed and pellet is resuspended in 5 mL chilled 0.1 M CaCl2
    • Suspension is incubated on ice for 10 min.
  • Suspensions are spinned down 5' @ 4000 rpm, 4 °C
    • Pellets were left on ice for undetermined time due to lack of liquid nitrogen
  • Supernatant is removed and pellet is resuspended in 2000 μL chilled 0.1 M CaCl2+10% glycerol prior to making aliquots.
  • Cells are divided in 50 μL aliquotes
  • Cells are stored @ -80 °C
OD600
Culture t = 1.5 h t = 2.0 h Quality
DB3.1(A)
0.098 0.172
DB3.1(B)
0.107 0.192 TODO
TOP10(A)
0.062 0.120
TOP10(B)
0.071 0.136 TODO

Miscellaneous

  • Something I finished

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