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5'-End cDNA Amplification Using Classic RACE Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4131

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "5'-end" partial cDNA clones using classic RACE, the first-strand products are generated by reverse transcription (primer extension) from a known gene-specific primer (GSP-RT). Then, a poly(A) tail is appended using terminal deoxynucleotidyltransferase (Tdt) and dATP. Amplification is carried out using three primers. - [Read 5'-End cDNA Amplification Using Classic RACE Protocol]

Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_63-65.pdf

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Differential Display-PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3844

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]

Indirect Peroxidase Technique Protocol - http://www.nottingham.ac.uk/pathology/protocols/indfroz.html

Category: Immunohistochemistry Protocols

Protocol for indirect peroxidase technique. Includes: Sections: Thickness-Between 5 and 10 microns. Pick up on chromic acid etched, poly-L-lysine or chrome alum/gelatine coated slides. Dry at room temperature overnight (not longer than 72 hours). - [Read Indirect Peroxidase Technique Protocol]

Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography - http://wheat.pw.usda.gov/~lazo/methods/au/m6e1.html

Category: RNA Protocols: mRNA Extraction Protocols

Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography. Total RNA is first isolated from the tissues or cells and then mRNA is isolated by PolyA+ selection using oligo(dT) cellulose. This is necessary for all tissue sources rich in RNase (and cell lines). Lazo Lab - [Read Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography]

Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography - http://wheat.pw.usda.gov/~lazo/methods/au/m6e1.html

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

Isolation of poly A+ RNA from Total RNA by oligo(deoxythymidine)cellulose Chromatography Protocol. Faulkner-Jones - [Read Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography]

POLY A+ RNA PREPARATION using proteinase K and SDS - http://wheat.pw.usda.gov/~lazo/methods/au/sect9.html

Category: RNA Protocols: mRNA Extraction Protocols

POLY A+ RNA PREPARATION using proteinase K and SDS. Bowtell Lab. - [Read POLY A+ RNA PREPARATION using proteinase K and SDS]

Poly-L-Lysine Coated Slides Protocol - http://www.nottingham.ac.uk/pathology/protocols/pll.html

Category: Immunohistochemistry Protocols: Coated Treated Slides Protocols

Protocol for poly-L-lysine coated slides. Includes: SKI STAINING MACHINE; SHANDON ELLIOT CAROUSEL. - [Read Poly-L-Lysine Coated Slides Protocol]

Purification of poly(A)+ RNA from total RNA - http://kuchem.kyoto-u.ac.jp/seika/shiraishi/protocols/oligotex.html

Category: RNA Protocols: mRNA Extraction Protocols

Purification of poly(A)+ RNA from total RNA. Hideaki Shiraishi, Kyoto University. The protocol employs Oligotex-dT30 for poly(A)+ RNA purification. - [Read Purification of poly(A)+ RNA from total RNA]

Random Primer Labeling of PolyA and RNA Protocol - http://molbiol.ru/eng/protocol/23_01.html

Category: Nucleic Acid Modification Protocols: Random Primer Labeling Protocols

Protocol for random primer labeling of poly A and RNA. Includes: Random primer reaction; cDNA purification; Hybridization; Washing; Stripping of membranes. - [Read Random Primer Labeling of PolyA and RNA Protocol]

Rapid Amplification of 3' cDNA Ends 3'-RACE Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3865

Category: PCR Protocols: cDNA Synthesis Protocols

3'-RACE reactions are used to isolate unknown 3' sequences or to map the 3' termini of mRNAs onto a gene sequence. 3'-RACE requires knowledge of a small region of sequence within either the target RNA or a partial clone of cDNA. A population of mRNAs is transcribed into cDNA with an adaptor-primer consisting at its 3' end of a poly(T) tract and at its 5' end of an arbitrary sequence of 30-40 nucleotides. - [Read Rapid Amplification of 3' cDNA Ends 3'-RACE Protocol]

Selection of Poly(A)+ RNA by Batch Chromatography - Subscription Required - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4048

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

When many RNA samples are to be processed or when working with small amounts (<50 Âµg) of total mammalian RNA, the technique of choice is batch chromatography on oligo(dT)-cellulose. The method described in this protocol uses a combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. Joseph Sambrook and David W. Russell. - [Read Selection of Poly(A)+ RNA by Batch Chromatography - Subscription Required]

Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography - Subscription Required - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4047

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

Chromatography on oligo(dT) columns is the preferred method for large-scale purification (>25 Âµg) 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]

Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3868

Category: DNA Protocols: cDNA Library Protocols

Protocol describes the generation of cDNA probes from poly(A)+ mRNA using random oligonucleotide primers. Probes of this type are used for differential screening of cDNA libraries. - [Read Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers Protocol]

Tubulin Polymerization with GTP/GMPCPP/Taxol Protocol - http://mitchison.med.harvard.edu/protocols/poly.html

Category: Cytoskeleton Protocols: Microtubules Protocols

Protocol for tubulin polymerization with GTP/GMPCPP/ Taxol. Includes: Solutions & Supplies; Prepolymerization Clarification; GTP Polymerization; Taxol Polymerization; GMPCPP Polymerization; Determining Concentration of GMPCPP/Taxol MTs. - [Read Tubulin Polymerization with GTP/GMPCPP/Taxol Protocol]

Yeast RNA Isolation: Large-Scale Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4154

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast RNA Isolation Protocols

Protocol describes large-scale yeast RNA isolation. It is designed to yield more than 10 mg of total nucleic acid and 300-400 µg of poly(A)-selected mRNA from each 500-ml culture. - [Read Yeast RNA Isolation: Large-Scale Protocol]

Articles (1-10 of 17)

Single Stranded Plasmid DNA Isolation Protocol

A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Drosophila Cell Transfection Protocol

A simple Drosophila tissue culture cell transfection method.

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Probe Preparation for 22 x 40 mm DNA Microarrays

Probe Preparation for 22 x 40 mm DNA Microarrays Protocol.

Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.