dependent Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR.  Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs - [Read Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

The recommended amount of RSV-ß-Galactosidase plasmid to use for transfection of cells (60 mm or 100 mm dish) is 1-2 µg. The optimal amount of plasmid DNA will be determined by the efficiency of transfection , which is very dependent upon the particular cell line and transfection protocol. - [Read B- Galactosidase Assay Protocol]

Category: Mouse Protocols

Protocol for calcium dependent by pentraxins to ligands immobilized on agarose, and effect of C-reactive protein on leptin action in mice in vivo. Includes: Testing for binding of CRP to immobilized leptin; Testing for binding of radiolabelled leptin by immobilized CRP, SAP and leptin receptor; Testing for effect of human CRP on human leptin action in mice. - [Read Calcium Dependent by Pentraxins to Ligands, and Effect of C-reactive Protein on Leptin Action]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol described here produces chromatin with regularly spaced nucleosomes having physiological nucleosome repeat lengths; something that can be difficult to achieve with purified components. In addition, chromatin assembled with the S-190 Chromatin Assembly Extract contains the ATP-dependent chromatin remodeling factors necessary for efficient transcription. - [Read Chromatin Assembly on Template DNA with Transcription Factors and Drosophila S-190 Chromatin Assembl]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

The potential cytotoxicity of compounds under hypoxic conditions is determined by exposing cell cultures to test compounds in a low oxygen atmosphere.  Subsequent cell survival is determined by the MTT and methylene blue colorimetric assays. - [Read Colorimetric Cytotoxcity Assays for Anchorage Dependent Cells Protocol]

Category: Immunology: T Cell Protocols

Protocol for NKT cells and CD8+ T cells are dispensable for T cell dependent allergic airway inflammation. - [Read NKT Cells and CD8+ T Cells are Dispensable for T Cell Dependent Allergic Airway Inflammation]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for preparation of messenger-dependent rabbit reticulocyte lysate. Informamtion also added to test that the lysate is working before doing the Nuclease inactivation - [Read Preparation of Messenger-Dependent Rabbit Reticulocyte Lysate]

Category: RNAi Technology Protocols

Short interfering RNAs (siRNAs) can be used to prime RNA synthesis by the RNA-dependent RNA polymerase (RdRP).  SiRNAs can be used by RdRP as primers for specific cellular mRNAs, forming dsRNA products capable of inducing transitive RNAi. - [Read Protocol for siRNA-Primed RNA Synthesis Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Small ubiquitin-like modifier (SUMO) is a small molecule, but has a variety of regulatory functions in cells. SUMO modification is involved in transcriptional regulation, subcellular localization, and protein-protein interactions. SUMO conjugation requires sequential E1-dependent activation, E2-dependent conjugation, and E3-dependent ligation steps. Protocol includes: In vivo and in vitro SUMOylation assay and deSUMOylation assay. - [Read Sumoylation and Desumoylation Assays for a Chromatin-Remodelling Complex In Vivo and In Vitro]