Molecular Biology
and Lysosomal Disease
Diagnosis Laboratory
Center for Research and Diagnosis of Genetic Diseases

RNA and DNA

Competent Bacteria

  • Inoculate bacterias E. coli (DH5, Top10', DM1) frozen at -80oC in 5ml of LB medium.
  • Incubate at 37oC overnight shaking at 170 rpm.
  • Add 0.5ml cell for 100 ml LB medium.
  • Incubate at 37oC, shaking at 160 rpm
  • Measure the OD600 every 30 min until reaching 0.5(must be between 0.4 e 0.6).

Tip: Never allow bacteria to grow too much. If the density was more than 0.65, culture is too dense and the efficiency of transformation will be reduced.

  • Cool the cells on ice for 20 min e centrifuge it at 4oC, 5000 rpm for 3 min.
  • Aspirate the medium.
  • Resuspend  cells in 50ml of sterile CaCI2 0.1M kept at 4OC.
  • Incubate cells on ice for 45 to 60 min.
  • Centrifuge at 2000 rpm for 5 min at 4ºC and gently to resuspend cells in 20 ml of cold sterile 0.1 M CaCl2.
  • Add sterile glycerol to the cell solution with gentle stirring to final concentration of 25% and 50% LB.
  • Aliquot the cells into tubes, freeze and store at -80 ° C.
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