• E. coli DH5alpha


  1. Take out competent E.coli cells from –80 C freezer.
  2. Turn on water bath to 42 C.
  3. Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar) – you may need more or less cells, depending how competent they are.
  4. Keep tubes on ice.
  5. Add 50 ng or 2.5 ul of circular DNA into E.coli cells. Incubate on ice for 30 min to thaw competent cells.
  6. Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
  7. Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
  8. Add 1 ml of SOC. Incubate tubes for 2 hours at 37 C.
  9. Spread about 100 ul of the resulting culture on LB plates with the appropriate antibiotic. Grow overnight.
  10. Pick the colonies about 12-16 hours later.