Stationary phase cells and aminopterin

These times refer to cells with a 24 h generation time. It is not readily applicable to cells with a short generation time in which t S is greater than half T. 

The double thymidine block is the method most frequently referred to in the literature but it is tedious to apply and suffers from the disadvantage that the cells enter S-phase with a pool of dTTP which is decreasing over the first hour or so and this makes estimation of 

The isoleucine/hydroxyurea method also involves two medium changes but the first of these may be replaced by simply adding isoleucine to the deficient medium. 

The stationary phase and aminopterin method suffers from the disadvantage that it takes a week to prepare the cells but thereafter it is straightforward and DNA synthesis can be followed without interference from endogenous pools ( 12.1.1). 

Variations and other combinations of these methods are obviously possible, e.g. mitotic selection and thymidine block aminopterin block reversed with low thymidine followed by a high thymidine block reversed with deoxycytidine. 

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