Enzymes and Buffers

Sequenase (version 2.0)

Sequenase is supplied by the manufacturer at a concentration of approx. 13 units/pl (approx. 1 mg/ml). Store at -20°C (not in a frost-free freezer).

Yeast inorganic pyrophosphatase

Optional, please see Step 6. This is supplied by the manufacturer (USB/Amersham Life Science) at a concentration of 5 units/ml; it catalyzes the hydrolysis of pyrophosphate to two molecules of orthophosphate. Pyrophosphatase can be mixed with Sequenase at a ratio of 1 unit of pyrophosphatase to 3 units of Sequenase. This is best done by mixing equal volumes of the two enzyme preparations and diluting the mixture with 6 volumes of enzyme dilution buffer. This dilutes Sequenase to its working concentration of approx. 1.6 units/pl and adds enough pyrophosphatase to prevent build-up of pyrophosphate; 2 pl of the mixture of diluted enzymes is required for each sequencing reaction.

Was this article helpful?

0 0

Post a comment