purpose 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: Cell Organelle Protocols: Mitochondria Protocols

The technique of JC-1 staining has been developed with the intent to detect DY in intact, viable cells. For this purpose JC-1 acts as a marker of mitochondrial activity, since the formation of J-aggregates, which give red emission, is reversible. Cells with high DY are those forming J-aggregates, thus showing high red fluorescence. On the other hand, cells with low DY are those in which JC-1 maintains (or re-acquire) monomeric form, thus showing only green fluorescence. - [Read Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1]

Category: Cell Culture Protocols

Information on the methods used to cultivate large amounts of cells for the purpose of obtaining an endogenous or recombinant product. - [Read Large-Scale Cell Culture Protocol]

Category: RNA Protocols: cDNA Libraries Library

Purpose: To transfer DNA from bacterial colonies to nylon (or nitrocellulose) membrane so that a library can be screened by hybridization. Jim Howe Donis Keller Lab - [Read Method: Transfer of DNA from Bacterial Colonies to Nylon Membrane or Nitrocellulose]

Category: Cell Biology and Cell Culture

This protocol focuses on the interactions between L-selectin expressed on neutrophils and PNAd coated onto the plastic surface. The main purpose of the flow chamber assay is to visualize and measure interactions between flowing cells expressing a given adhesion molecule on their surface, and their receptor, either directly coated on the flow chamber lower wall or expressed on a cell monolayer. - [Read Protocol for L-selectin-PNAd Interactions under Flow Conditions.]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

Chromatography on oligo(dT) columns is the preferred method for large-scale purification (>25 µg) of poly(A)+ RNA extracted from mammalian cells. Typically, between 1% and 10% of the RNA applied to the oligo(dT) column is recovered as poly(A)+ RNA. Because the method can be frustratingly slow, it is not recommended for purification of poly(A)+ RNA from multiple samples. For this purpose, batch elution (Selection of Poly(A)+ RNA by Batch Chromatography) is the better choice. - [Read Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography - Subscription Required]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols

Protocol for total RNA isolation. This method is fast and works well for preparation of total RNA.  We typically use total RNA for quantitative S1 nuclease analysis of endogenous (or in vitro) gene expression, and this method works well for this purpose. - [Read Total RNA isolation (Guanidinium Thiocyanate-Phenol-Chloroform Extraction) Protocol]