synthetic Protocols

Category: Cloning Protocols

Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. - [Read Attaching Adaptors to Protruding Termini Protocol]

Category: DNA Protocols: cDNA Library Protocols

This stage achieves four goals: polishing the ends of double-stranded DNA, ligation of synthetic linkers or adaptors, digestion of the attached linkers to create cohesive termini, and preparing the cDNA for cloning. - [Read Attachment of Linkers or Adaptors for Construction of cDNA Libraries]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor.  This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest.  Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out. - [Read Competitive RT-PCR: Preparation of Competitor RNA Protocol]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

Complex yeast media protocols. Including: YPAD Medium or Also Called YPD plus Adenine; Synthetic Complete Drop Out (SC drop-out)Medium; Synthetic Complete drop-out Medium Mix (SC drop-out). - [Read Complex Yeast Media Protocols]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Includes staining protocols: Triple-Staining Adherent Cells with MitoTracker, Phalloidin,& Nuclear Dyes; Staining Adherent Cells with Cytokeratin Primary Antibodies & Synthetic Fluorophores; Staining Adherent Cells with Intermediate Filament Primary Antibodies & Synthetic Fluorophores; Staining Cells & Tissue Cryosections with Tubulin Primary Antibodies, Phallotoxins,& Synthetic Fluorophores; Triple-Staining Tissue Cryosections with Wheat Germ Agglutinin, Phalloidin,& Nuclear Dyes; etc. - [Read Confocal Microscopy Specimen Preparation Using Synthetic Fluorophores and Immunofluorescence]

Category: PCR Protocols

Method uses PCR to amplify and display many cDNAs derived from the mRNAs of a given cell or tissue type.  The method relies on two different types of synthetic oligonucleotides: anchored antisense primers and arbitrary sense primers.  A typical anchored primer is complementary to approx. 13 nucleotides of the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. - [Read Differential Display-PCR Protocol]

Category: Antibody Protocols: Epitope Mapping Antibody Protocols

A set of overlapping synthetic peptides is synthesized, each corresponding to a small segment of the linear sequence of a protein antigen and arrayed on a solid phase. The panel of solid-phase peptides is then probed with a test antibody, and bound antibody is detected using an enzyme-labeled secondary antibody. This method is very rapid and can be extraordinarily successful. - [Read Epitope Mapping Using Synthetic Biotin-Labeled Peptides Protocol]

Category: Cell Biology and Cell Culture

Cell fractionation of cellular components using Percoll a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. Percoll becomes a simple matter to establish a linear density gradient. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients. - [Read Equilibrium Density Gradient Percoll Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Method assesses cellular mRNA transcripts in tissue sections and cell cultures using unique short anti-sense primers directed against sequences in particular protein(s). The unlabeled synthetic cDNA oligonucleotide primers are extended complementary to a sense mRNA transcript using reverse transcriptase and labeled through incorporation of a fluorescent-labeled dUTP nucleotide base. This procedure provides rapid detection of low abundance mRNA messages that can be related to other cellular.... - [Read Fluorescent In Situ Transcription in Cells and Tissues Protocol]

Category: Antibody Protocols: Antibody Production Protocols

Novel strategy of immunizing a phosphorylated peptide or a synthetic phosphopeptide, which corresponds to the protein phosphorylated at a targeted residue. Method has been applied to the production of antibodies that can specifically recognize the other types of site-specific protein modification, such as acetylation, methylation, and proteolysis. - [Read Functional Analyses for Site-Specific Phosphorylation of a Target Protein in Cells]

Category: C. elegans Protocols: C. elegans Genetics Protocols

A guide to genetic mapping in C. elegans. Includes: Introduction and basics; Two-point mapping; Three-point mapping; Deficiency and duplication mapping; Basics of SNP mapping; Mapping dominant mutations; Mapping suppressor/enhance mutations; Mapping of synthetic mutations. - [Read Guide to Genetic Mapping in C. elegans]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Live-cell imaging techniques provide critical insight into the fundamental nature of cellular & tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein & synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology labs. Includes: Maintaining Live Cells on the Microscope Stage; Live-Cell Imaging Culture Chambers; Optical System and Detector Requirements etc. - [Read Introduction to Live-Cell Imaging Techniques]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Live-cell imaging techniques provide a critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology laboratories. - [Read Maintaining Live Cells on the Microscope Stage]

Category: Peptide Protocols: Antibodies from Peptides Protocols

Protocol for Antibody Creation from a Synthetic Peptide. Antibody from Peptides. Daniel O'Connor. - [Read Making antibodies to synthetic peptides]

Category: Antibody Protocols: Antibody Production Protocols

Protocol for making antibodies to synthetic peptides. - [Read Making Antibodies to Synthetic Peptides Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

Green fluorescent protein is commonly used to monitor gene expression and protein trafficking within intact cells. The Monster Green® Fluorescent Protein is encoded by an improved synthetic version of the green fluorescent protein gene originally cloned from Montastrea cavernosa (Great Star Coral). - [Read Monster Green® Fluorescent Protein Assay]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol details the purification and analysis of many synthetic peptides of 2-65 amino acid residues.  These peptides contain a number of ionizable or polar side chains, but do not contain secondary structural elements (such as ß-sheets) that favor supramolecular assembly. - [Read Purification of Peptides from Solid-Phase Peptide Synthesis with RP-HPLC Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Polyacrylamide Gels Protocols

Protocol for purification of synthetic oligonucleotides by polyacrylamide gel electrophoresis. As a rule of thumb, oligonucleotides >25 nucleotides should be purified by polyacrylamide gel electrophoresis, as should oligonucleotides of any length that yield anomalous results.  After electrophoresis, the oligonucleotide is eluted from the gel and concentrated by reversed-phase chromatography on Sep-Pak C18 columns. - [Read Purification of Synthetic Oligonucleotides by Polyacrylamide Gel Electrophoresis Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

A synthetic oligonucleotide annealed to single-stranded DNA derived from a recombinant bacteriophage M13 or phagemid template is used to prime the synthesis of complementary radiolabeled DNA. Synthesis is catalyzed by the Klenow fragment of E. coli DNA polymerase I, which extends the annealed primer for various distances along the single-stranded template DNA. - [Read Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates Protocol]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

PCR is used as a preparative tool for the synthesis of a high-complexity double-stranded DNA library. In the example presented here, a mixture of synthetic oligonucleotides is used to synthesize a random peptide NNK library, where K is either T or G. The exclusion of A and C nucleotides at the third position decreases the occurrence of stop codons but still allows codons for all 20 amino acids. - [Read Use of PCR to Prepare a Double-Stranded DNA Library Encoding Random Peptides Protocol]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

Yeast Media, Solutions and Stocks Protocols. Includes: Yeast Media; AHC Medium; AHC plates; SD Medium; SD plates; Sorbitol-Agar Medium; Sorbitol-Agar Medium Top Agar; Sporulation Medium Plates; YPD Medium; YPD plates; Synthetic Media Supplements. - [Read Yeast Media, Solutions and Stocks Protocols]