( mplate DNA in all optimization reaction...)Two-dimensional Optimization of a Semi-nested PCR,,, for Detecting Listeria monocytogenes

mplate DNA in all optimization reactions. The MgCl2 concentration as well as the annealing temperature were varied systematically. Amplified products were detectable on the agarose gel up to a temperature of 62.8C. The optimal annealing temperature should be below this temperature. At a MgCl2 concentration of 3 mM, the product yields were at their maximum; no increase in yield was ascertained when the concentration was increased to 3.5 mM.

Second optimization step: MgCl2 concentration 3 mM; temperature gradient: 55C 65C (Fig. 3)

In order to further determine the exact annealing temperature, a temperature window of 55C 65C was applied at the optimal MgCl2 concentration of 3 mM. A visual assessment of the band intensity was sufficient for assuming a temperature optimum of 58C.

To quantify the product yield, a densitometric measurement of the electrophoretically separated amplification products was carried out.

Fig.1. Determining the detection limit for Listeria monocytogenes DNA based on the initial PCR (primer pair L1/L4), as well as on the subsequent semi-nested PCR (primer pair L2/L4). Starting conditions: 20 pg 2 fg DNA per reaction; 1.5 mM MgCl2; 55C annealing temperature. Densitometric measurement (Fig. 4)

Using an OD measurement, the relative product yield is determined on the basis of the intensity of the bands in the gel (in Fig. 3). With rising temperature, the OD also increased continuously in the initial r
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