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G1/S cell synchronization using double thymidine block. This protocol is designed to synchronize cells at the G1/S border (i.e G1 phase). It is highly effect and gives almost complete synchronization in the culture. Note that this protocol will not work on cell lines with intact p53 apoptotic responses.


Procedure works best with p53 deficient cells (ex. HeLa and U2-OS)

Thymidine MW 242.23 - for 200mM stock (100X) add 0.484g in 10ml



  1. Grow HeLa cells in standard DMEM +10%FBS to about 40% confluencey.
  2. Simply add thymidine that has been resuspended in PBS to a final concentration of 2mM in the media. (for 200mM stock add 0.484g in 10mls)
  3. Incubate culture at 37C for 19hrs (strict time keeping here).
  4. Remove media and wash cells with PBS 3X.
  5. Add fresh media without thymidine and let incubate for 9 hrs at 37C.
  6. Add thymidine again to a final of 2mM and allow to incubate for another 16hrs.
  7. Wash cells with PBS and add fresh media. The cells are now in G1 and will be "released" to progress through the cell cycle over the next 15-20hrs. The cells should be uniform for about 1-2 cell divisions and then regain their asynchronous state.



  1. This procedure is designed for p53 deficient cells.
  2. Timing is essential. Typically cells are harvested in 2 or 3 hr intervals over the next 15-24hrs and nocodazole can be added to prevent progression through mitosis.


Relevant papers and books

  1. [None]


  • Jeremy Myers

--Jeremy S. Myers 15:48, 11 April 2008 (EDT) or instead, discuss this protocol.

Post questions or comments on my talk