Sequenase reaction buffer with MnCl₂

25 mM MnCl₂

125 mM NaCl

200 mM Tris-Cl (pH 7.5)

For a 5x buffer. Store at -20°C.

The presence of Mn²⁺ in the Sequenase reaction buffer increases the efficiency with which Sequenase will utilize ddNTPs, even if Mg²⁺ is also present. The net result is a more efficient termination of sequencing reactions closer to the starting primer. This modification is useful for improving the strength of the bands that run close to the oligonucleotide primer or when using a base analog such as dITP to iron out compressions in GC-rich regions of sequence.

To use Mn²⁺, add 1 μl of 5x Sequenase reaction buffer with MnCl₂ to the labeling reaction before adding Sequenase. Alternatively, add 0.01 volume of 1 M MnCl₂ to the dideoxy-dNTP extension/termination mixtures. Do not use manganese-containing buffers that develop a slight yellowish color or that contain a yellow-brown precipitate.