Abstract: Silica-coated magnetic nanoparticles with greater ability to remain dispersed, and methods of making and using silica-coated magnetic nanoparticles. The magnetic nanoparticles comprise a core and a coating, where the core comprises Fe3O4 or other magnetic material and the coating has a thickness of from about 1.5 nm to about 2 nm. The magnetic nanoparticles are useful for preparing nucleic acids for analysis, by separating nucleic acids from other components and by normalizing nucleic acid concentrations.

Abstract: Methods and compositions are provided for amplifying a pool of oligonucleotides, such as dual guide oligonucleotide constructs comprising sequences encoding a first guide RNA segment and a sequence encoding a second guide RNA segment. An amplification mixture is formed comprising the pool of oligonucleotides, an amplification enzyme, deoxyribonucleotide triphosphates, and primers. The amplification mixture is thermocycled a sufficient number of times and under conditions to produce a library of oligonucleotide constructs. The present methods and compositions provide dual guide libraries, including libraries that are essentially free of scrambled library members.

Abstract: Provided herein is a method for fabricating transformable or transfectable molecules that includes an assembly reaction containing a variety of pre-made cassettes possessing ends that hybridize to one another, transforming or transfecting said molecules into a desired host cell and then selecting a transformed/transfected host cell containing plasmid molecules composed of said the cassettes. A kit for performing the method is also provided.

Abstract: Provided herein is a method for fabricating transformable or transfectable molecules that includes an assembly reaction containing a variety of pre-made cassettes possessing ends that hybridize to one another, transforming or transfecting said molecules into a desired host cell and then selecting a transformed/transfected host cell containing plasmid molecules composed of said the cassettes. A kit for performing the method is also provided.

Abstract: Provided herein is a method for fabricating transformable or transfectable molecules that includes an assembly reaction containing a variety of pre-made cassettes possessing ends that hybridize to one another, transforming or transfecting said molecules into a desired host cell and then selecting a transformed/transfected host cell containing plasmid molecules composed of said the cassettes. A kit for performing the method is also provided.

Abstract: Methods and compositions are provided for amplifying a pool of oligonucleotides, such as dual guide oligonucleotide constructs comprising sequences encoding a first guide RNA segment and a sequence encoding a second guide RNA segment. An amplification mixture is formed comprising the pool of oligonucleotides, an amplification enzyme, deoxyribonucleotide triphosphates, and primers. The amplification mixture is thermocycled a sufficient number of times and under conditions to produce a library of oligonucleotide constructs. The present methods and compositions provide dual guide libraries, including libraries that are essentially free of scrambled library members.

Abstract: Provided herein is a reaction mixture comprising Cas9 and a non-ionic surfactant, e.g., a polyoxyethylene surfactant. In certain embodiments, the reaction mixture may comprise a Cas9 protein, a guide RNA, a salt, a buffering agent, a nucleic acid target and a non-ionic surfactant. Kits are also provided. In certain embodiments, a kit may comprise: a Cas9 protein; and a concentrated reaction buffer comprising salt, a buffering agent and a non-ionic surfactant.

Abstract: Provided herein is a method for fabricating transformable or transfectable molecules that includes an assembly reaction containing a variety of pre-made cassettes possessing ends that hybridize to one another, transforming or transfecting said molecules into a desired host cell and then selecting a transformed/transfected host cell containing plasmid molecules composed of said the cassettes. A kit for performing the method is also provided.

Abstract: Provided herein is a reaction mixture comprising Cas9 and a non-ionic surfactant, e.g., a polyoxyethylene surfactant. In certain embodiments, the reaction mixture may comprise a Cas9 protein, a guide RNA, a salt, a buffering agent, a nucleic acid target and a non-ionic surfactant. Kits are also provided. In certain embodiments, a kit may comprise: a Cas9 protein; and a concentrated reaction buffer comprising salt, a buffering agent and a non-ionic surfactant.

Abstract: The invention provides for polynucleotides and vectors comprising at least two tag sequences. In particular, preferred vectors are viral vectors. The invention also provides for polynucleotides and vectors comprising a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for polynucleotides and vectors wherein a gene of interest is fused in frame to at least two tag sequences, for example, a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for methods of using the polynucleotides and vectors of the invention for detecting and/isolating protein complexes or identifying a binding partner for a protein of interest.

Abstract: The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed.

Abstract: The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed.

Abstract: The invention relates to acyclic chain terminator nucleotide analogs. More particularly, the invention relates to phosphonomethoxyethyl nucleotide analogs and detectably labeled versions thereof, especially fluorescently labeled versions thereof. The invention further relates to the use of chain terminating phosphonomethoxyethyl nucleotide analogs in methods of synthesizing a polynucleotide, labeling a polynucleotide, determining polynucleotide sequence information, and kits therefor.

Abstract: The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed.

Abstract: The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed.

Abstract: The invention relates to fluorescent dyes. More particularly, the invention relates to fluorescent cyanine dyes, and especially to water soluble fluorescent cyanine dyes that contain additional sites for attachment to biomolecules. The invention provides a group of novel, water soluble fluorescent cyanine dyes that have distinct fluorescence characteristics that permit their use in any assay or method suited to water soluble fluorescent dyes, and especially to assays requiring a plurality of distinguishable fluorescent markers. The invention further relates to nucleotides, nucleosides, polynucleotides and polypeptides labeled with novel fluorescent cyanine dyes according to the invention, and methods of using them.

Abstract: The present invention relates to a dual-labeled nucleotide comprising a fluorescent label and a quencher of that fluorescent label, wherein the quencher is attached to a phosphate moiety that is cleaved off when the nucleotide is enzymatically incorporated into a polynucleotide. The invention further relates to methods of labeling a polynucleotide molecule, as well as methods for identifying one or more residues of a polynucleotide using the dual-labeled nucleotide analogs of the present invention and kits comprising dual-labeled nucleotide analogs according to the invention.

Abstract: The invention relates to fluorescent dyes. More particularly, the invention relates to fluorescent cyanine dyes, and especially to water soluble fluorescent cyanine dyes that contain additional sites for attachment to biomolecules. The invention provides a group of novel, water soluble fluorescent cyanine dyes that have distinct fluorescence characteristics that permit their use in any assay or method suited to water soluble fluorescent dyes, and especially to assays requiring a plurality of distinguishable fluorescent markers. The invention further relates to nucleotides, nucleosides, polynucleotides and polypeptides labeled with novel fluorescent cyanine dyes according to the invention, and methods of using them.

Abstract: The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first, and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed.

Abstract: The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed.