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Competent cells with Hanahan's method - (Sep/22/2014 )


I am new to this forum and new to molecular cloning.


I'm working on a project that require me to prepare new competent cells - TOP10.

I am a bit confused at STEP 8 and 9  with I followed the protocol :


  1. Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
  2. Centrifuge at 3000rpm at 4°C for 10 minutes in a flat bottom centrifuge bottle.
  3. Discard supernatant by pouring out slowly and pipeting remaining supernatent
  4. Gently resuspend in 80 ml of ice cold CCMB80 buffer
  5. Incubate on ice 20 minutes
  6. Centrifuge again at 4°C and discard supernatant as described above.
  7. Resuspend in 10 ml of ice cold CCMB80 buffer.
  8. Test OD of a mixture of 200 μl SOB and 50 μl of the resuspended cells.
  9. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
  10. Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
  11. Store at -80°C indefinitely.
  12. Test competence (see below)
  13. Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles

Do I have to adjust the rest of the suspension from step 7 with SOB and take OD reading and then readjust again with CCMB80?

If not, then I don't see the point as to why I would need to adjust with SOB and then adjust with CCMB80, and why I couldn't just adjust with CCMB80 in the first place.


Any suggestion would be greatly appreciate.  :)


Step 8 is intended to measure the OD in the context of the SOB medium, which is similar to the original growth medium. This allows a comparison to be made with the original culture density. In step 9, you dilute just with CCMB80, not with SOB. This step is not all that critical, but it does increase consistency of the resulting cells.


Thank you for clearing that up.  I really appreciate your help! :)