The present disclosure provides methods, compositions, and systems for distributing polymerase compositions into array regions. In particular, the described methods, compositions, and systems utilize density differentials and/or additives to increase efficiency in the distribution of polymerase compositions to a surface as compared to methods utilizing only diffusion control.

The present disclosure provides methods, compositions, and systems for distributing polymerase compositions into array regions. In particular, the described methods, compositions, and systems utilize density differentials and/or additives to increase efficiency in the distribution of polymerase compositions to a surface as compared to methods utilizing only diffusion control.

Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.

Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.

In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of at least one agent that generates a ligatable terminal 5′ phosphate group by removing an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, an aprataxin enzyme can catalyze removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of an aprataxin enzyme under conditions suitable for ligating nucleic acid ends.

In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of at least one agent that generates a ligatable terminal 5′ phosphate group by removing an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, an aprataxin enzyme can catalyze removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of an aprataxin enzyme under conditions suitable for ligating nucleic acid ends.

Provided herein is a reverse transcriptase mixture comprising a reverse transcriptase and a colored dye at a concentration in the range of 0.003%-1% (v/w). The colored dye may be visually observed during transfer of the mix from one vessel to another and addition of the mix to another mix can be confirmed by eye by observing the colored dye.

A reaction vessel comprises a lower chamber with a first volume, and an upper chamber with a second volume greater than the first volume. A thermocycling system for heating the reaction vessel includes a lower heating zone to heat the lower chamber, an upper heating zone to heat the upper chamber, and a lid heater to heat an opening of the upper chamber. A method comprises loading a sample into a lower chamber of a reaction vessel, thermocycling the lower chamber using a lower heating zone of the thermo cycler, combining an additive into the sample to produce a combination filling the lower chamber and at least partially filling an upper chamber of the reaction vessel, and incubating the upper and lower chambers using the lower heating zone and an upper heating zone. The lower and upper chambers can have different wall thicknesses to facilitate heat transfer.

A reaction vessel comprises a lower chamber with a first volume, and an upper chamber with a second volume greater than the first volume. A thermocycling system for heating the reaction vessel includes a lower heating zone to heat the lower chamber, an upper heating zone to heat the upper chamber, and a lid heater to heat an opening of the upper chamber. A method comprises loading a sample into a lower chamber of a reaction vessel, thermocycling the lower chamber using a lower heating zone of the thermo cycler, combining an additive into the sample to produce a combination filling the lower chamber and at least partially filling an upper chamber of the reaction vessel, and incubating the upper and lower chambers using the lower heating zone and an upper heating zone. The lower and upper chambers can have different wall thicknesses to facilitate heat transfer.

Provided herein is a composition comprising an enzyme that releases pyrophosphate from a substrate and a dye of Formula 1. A method for detecting pyrophosphate is also provided. A kit comprising a polymerase that releases pyrophosphate by hydrolysis of nucleoside triphosphates during nucleic acid replication, a divalent manganese salt, and the dye are also provided. The present composition, method and kits provide a way to detect and/or quantify substrates or products of enzyme reacted substrates associated with the release pyrophosphate (e.g., nucleic acid amplification reactions and other reactions that hydrolyze ATP) via a distinct color change without substantially affecting the sensitivity and/or specificity of the reaction.