Although TE is often used to dissolve primers and to resuspend templates, the presence of excess EDTA can result in poor sequencing results. In this experiment the concentration of EDTA was varies in a terminator reaction by adding difference amounts of TE. The amount used was pGem 3Zf(+) and the primer was the –21 M13 forward primer.

1 x TE: 10mM Tris-C1, pH 8.0, 1mM EDTA
10 x TE: 100mM tris-C1, pH 8.0, 10mM EDTA

EDTA cheletes two molecules of Mg/EDTA molecule

Conclusion:

The reaction will tolerate as much as .25mM EDTA without resulting in too much loss of signal. This reduces the amount of Mg in the reaction to 1.5mM. With .5mM EDTA (1.0mM Mg) the signal strength has been reduces by 75% and the reaction dies out earlier than normal. If more than .25mM EDTA will be added to the reaction from templates or primers, add sufficient Mg to restore the concentration of free Mg to above 1.5mM.

*This reaction was essentially dead.