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(LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4107

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols: Tissue Preparation for LCM Protocols

LCM isolates specific cells or tissues from samples mounted on microscope slides. The samples are viewed through a thermoplastic film that is attached to a microcentrifuge tube lid. Localized heat, caused by the application of a laser pulse, fuses the membrane to the cells of interest, which can then be harvested for further analysis. RNA and proteins can be purified from the isolated cells, allowing detailed analysis of gene expression. This protocol is divided into three stages. - [Read (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel]

Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4159

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

In the method described here, the cells are lysed by a simple freeze/thaw protocol and . . . - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes]

Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4159

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

In this protocol the cells are lysed by a simple freeze/thaw protocol. - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes]

Concentration of Oligo DNA by Ethanol Precipitation Protocol - http://mycoplasmas.vm.iastate.edu/lab_site/methods/DNA/precip.html

Category: Oligonucleotide Protocols: Oligonucleotide Purification Protocols

Concentration of DNA by Ethanol Precipitation Protocol. Adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma. Usually 2.5 - 3 volumes of ethanol and/or acetate solution is added to the DNA in a microcentrifuge tube. This is then put into an ice-water bath for at least 10 minutes. The precipitation is performed by incubation at -20C overnight. - [Read Concentration of Oligo DNA by Ethanol Precipitation Protocol]

Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4456

Category: Nucleic Acid Purification Protocols

Protocol describes the standard method to recover nucleic acids from aqueous solutions by precipitation of DNA with ethanol. Subnanogram amounts of DNA (and RNA) can be quantitatively precipitated with ethanol, collected by centrifugation, and redissolved within minutes. - [Read Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes Protocol]

Articles (1-8 of 8)

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.

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.

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.

DNA Ligation Protocol

The DNA Ligation protocol described here contains the steps required to join together using ligase enzyme both plasmid DNA and insert DNA fragments in order to create a new plasmid. This new ligated plasmid can be transformed after into competent bacteria to produce DNA for mini, midi or maxi-prep isolation.

Histone H1 Kinase Activity Assay

Histone H1 Kinase Activity Assay Protocol. This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.

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