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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]

A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower - http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb16/16183-2.pdf

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

The protocol includes: organelle isolation, deoxyribonuclease treatment, lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple samples of organelle DNA from a small amount of tissue. - [Read A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower]

A Method for Micro-Scale Isolation and Purification of Gangliosides - http://www.glycotech.com/protocols/Proto1.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

Solvent partition protocol allows the isolation of gangliosides from small samples and from samples where ganglioside concentrations are low, especially relative to the concentration of potentially contaminating proteins and other large molecular weight species. Stephan Ladisch Director, Center for Cancer and Transplant Biology, Children's National Medical Center, Washington, D.C. - [Read A Method for Micro-Scale Isolation and Purification of Gangliosides]

A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4117

Category: Genetics and Genomics: Cancer Genetics Protocols: Loss of Heterozygosity Protocols

DNA for analysis is purified using salt precipitation. The method is gentle, limits the breakage of the long chromosomal strands, and avoids the use of phenol and chloroform. It is suitable for use with cultured cells, breast tumor tissue that has been subjected to hormone receptor analysis, and blood samples. The loss of heterozygosity assay is performed using a multiplex PCR, in which one of each primer pair is labeled with a different fluorophor. - [Read A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome]

Agarose Gel Electrophoresis PDF - http://www.uni-tuebingen.de/evoeco/files/methods/Electrophoresis.pdf

Category: Molecular Biology Protocols: Agarose Gel Preparation

Agarose Gel Electrophoresis. Pouring of Agarose Gel, Preparation of samples, Gel electrophoresis, Ethidium bromide staining. - [Read Agarose Gel Electrophoresis PDF]

An Alternatively Spliced Isoform of t(8;21) Transcript Promotes Leukemogenesis - http://www.natureprotocols.com/2006/08/03/an_alternatively_spliced_isofo.php

Category: Genetics and Genomics: Cancer Genetics Protocols

Describe the methods to identify and quantitate the specific A/E9a transcript in t(8;21) patients samples relative to the AML1-ETO transcript encoding the well known full-length 752 amino acid AML1-ETO protein (AE). Includes: RNA preparation and RT-PCR; Relative quantitation of the AE9a and the AE transcripts. - [Read An Alternatively Spliced Isoform of t(8;21) Transcript Promotes Leukemogenesis]

Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required) - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4432

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Analysis of DNA Fragmentation Using the JAM Assay. By Shailaja Kasibhatla et al., The JAM assay is based on labeling nuclear DNA of cycling cells with [3H]thymidine and harvesting samples on glass fiber filters. Apoptosis will generate DNA fragments small enough to pass through the glass fiber filter, resulting in decreased radioactivity of the particular sample. Cell-mediated cytotoxicity or cell killing mediated by cytotoxic T lymphocytes (CTL) can also be measured by this technique. - [Read Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required)]

Analysis of DNA Methylation using Bisulphite Sequencing Protocol - http://www.methods.info/Methods/DNA_methylation/Bisulphite_sequencing.html

Category: PCR Protocols: Bisulfite Conversion Based PCR Protocols

Protocol for the analysis of DNA methylation using bisulphite sequencing. Method allows precise analysis of methylation in a certain region by converting all nonmethylated cytosines into tymines, while methylated cytosines remain unchanged. This method requires small amount of genomic DNA and therefore seems to be very useful for the analysis of clinical samples, where the material amount is limited.
- [Read Analysis of DNA Methylation using Bisulphite Sequencing Protocol]

Apoptosis Detection Protocol By Single Laser Flow Cytometry - http://cyto.mednet.ucla.edu/Protocols/detection.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A sensitive method for the detection of apoptosis by single laser flow cytometry. Methodology includes: Staining for detection of apoptosis, Direct Staining Procedure, Indirect Staining Procedure, Protocol for the use of actinomycin D (AD) on samples that were stained with 7-AAD for apoptosis and fixed in formaldehyde. - [Read Apoptosis Detection Protocol By Single Laser Flow Cytometry]

Autoradiography Protocols - http://pingu.salk.edu/~sefton/Hyper_protocols/ARG.html

Category: Radioactivity: Autoradiography

Autoradiography Protocols. Exposing gels and plates containing radioactive samples to X-ray film. Detection of 3H. Detection of 35S and 14C. Detection of 32P and 125I. Bart's Cookbook. - [Read Autoradiography Protocols]

Carbohydrate Assays - http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=13

Category: Molecular Biology Protocols: Carbohydrate Protocols

o determine the relative amounts of LPS carbohydrates present in a given strain. The assay can be done on one set of samples and then scanned at the various wavelengths for reasonable data on the 3 types of sugars. HEXOSE ASSAY, 6-DEOXYHEXOSE ASSAY, HEPTOSE ASSAY. Hancock Laboratory. - [Read Carbohydrate Assays]

CGH of Direct Labeled Test DNA vs Normal DNA Protocol - http://waldman.ucsf.edu/Protocols/directcgh.html

Category: Cytogenetics Protocols

This CGH Protocol is used for DNA of good quality when available in sufficient amounts. We usually do replicate hybridizations using samples labeled "inversely" (reversing the label for test and normal DNA's). If appreciable artifact occurs, then alternative labels are tried. - [Read CGH of Direct Labeled Test DNA vs Normal DNA Protocol]

Clonality - X Chromosome Inactivation Assay Protocol - http://cgap-mf.nih.gov/Protocols/DNARNAProteomicAnalysis/DNA/DNAProtocols/Clonality.html

Category: Genetics and Genomics: Cancer Genetics Protocols

Investigators can utilize X chromosome inactivation (methylation) to determine the clonality status of a tumor or premalignant lesion in females. The technique is based on a methylation-sensitive restriction enzyme and analysis of a polymorphic locus on the X chromosome. Clonal cell populations will show "loss" of the non-methylated allele after restriction digest. The assay can be performed on DNA recovered from microdissected samples. Both frozen tissue and fixed-embedded tissue can be used. - [Read Clonality - X Chromosome Inactivation Assay Protocol]

Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4350

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Protocol provides methods for cryofreezing and subsequent thawing of mammalian cells. Pre-confluent cells are trypsinized, pelleted, resuspended in freezing medium, and gradually frozen. When needed, frozen cells are thawed quickly under running tap water and transferred to growth medium. - [Read Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol]

CYP1A1-Inducing Potency and Cytotoxicity Test in the HEPA-1 Mouse Hepatoma Cell Line - http://embryo.ib.amwaw.edu.pl/invittox/prot/112.htm

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

This bioassay utilizes cultured Hepa-lclc7 (Hepa-1) mouse hepatoma cells to assess the CYPlA1-inducing potency or cytotoxicity of pure test chemicals or environmental samples. In the Hepa-l induction test , the CYPlA1-inducing potency of the test sample is detected as increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD) activities. - [Read CYP1A1-Inducing Potency and Cytotoxicity Test in the HEPA-1 Mouse Hepatoma Cell Line]

Cytokine ELISA Protocol - http://www.bdbiosciences.com/pharmingen/protocols/Cytokine_ELISA.shtml

Category: Signalling Signal Transduction Protocols: Cytokine Protocols

Capture antibody, Blocking, Standards and Samples, Detection antibody, Cytokine ELISA Helpful Hints. BD Biosciences. - [Read Cytokine ELISA Protocol]

Dealing with Carryover Contamination in PCR: An Enzymatic Strategy Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4092

Category: PCR Protocols

Protocol for dealing with carryover contamination in PCR- enzymatic strategy. Repeated use of PCR and manipulation of its products cause aerosols that can contaminate neighboring samples and work areas. Such "carryover contamination" can be prevented by including dUTP in place of dTTP for all amplification reactions. - [Read Dealing with Carryover Contamination in PCR: An Enzymatic Strategy Protocol]

Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples - http://www.ambion.com/techlib/tn/131/6.html

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Three Ambion kits were used to quantitate specific miRNAs and to detect differential miRNA expression in various mouse brain regions and cell types isolated by laser capture microdissection (LCM). These techniques can be applied to studying miRNA in other species, tissues, and cell types. Includes: Obtain Laser Capture Microdissected Samples; Isolate miRNA from LCM Samples; Quantitate miRNA by qRT-PCR. - [Read Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples]

Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples - http://www.ambion.com/techlib/tn/131/6.html

Category: RNAi Technology Protocols

Information on how detect and quantitate MicroRNA in laser capture microdissection samples. Includes: Obtain Laser Capture Microdissected Samples; Isolate miRNA from LCM Samples; Quantitate miRNA by qRT-PCR; Detection of miRNA in Microdissected Tissue from Mouse Brain by qRT-PCR; Differential Expression of MicroRNA in Whole Brain Tissue Compared to a More Homogeneous Population of Cells. - [Read Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples]

Detection of Autoantibodies with Self-Assembling Radiolabeled Antigen Tetramers Protocol - http://www.natureprotocols.com/2007/01/29/detection_of_autoantibodies_wi.php

Category: Immunology

Protocol for detection of autoantibodies with self-assembling radiolabeled antigen tetramers. Details how to produce radiolabeled antigen-streptavidin tetramers for detection of antibodies by immunoprecipitation. Optionally, the antigen tetramers can be denatured to compare responses to folded and unfolded antigen in the same system. This technique can be applied to a large or small number of samples, and a given sample can be simultaneously assayed with multiple antigens. - [Read Detection of Autoantibodies with Self-Assembling Radiolabeled Antigen Tetramers Protocol]

Detection of Fungi by Fluorescence Microscopy Using Fluorescent Brighteners - http://www.aspergillus.org.uk/indexhome.htm?secure/laboratory_protocols/./flbr.html~main

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Certain fluorescent dyes such as Blankophor have a high affinity for the b -glycosidically linked polysaccharides such as glucan and chitin, which are main the constituents of the fungal cell wall. Therefore, these fluorescent dyes can be used for screening clinical samples for the presence of fungal elements. This procedure can be performed using the following specimens: Nail, Skin, Bronchial alveolar lavage fluid, Sputum and Biopsies. - [Read Detection of Fungi by Fluorescence Microscopy Using Fluorescent Brighteners]

DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4098

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

In this protocol, the DNA-binding capacity of Wizard MagneSil particles is used to capture and release a consistent amount of DNA (100 ng) across a wide range of samples. At the end of the procedure, the DNA is eluted into 100 µl Elution Buffer to give a final concentration of 1 ng/µl, relieving the need for postpurification DNA quantitation. - [Read DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs Protocol]

DNA laddering assay for treated cells - http://www.celldeath.de/apometh/dnaladd.html

Category: Cell Biology and Cell Culture: Apoptosis Protocols

DNA laddering can be detected from samples with only 8% apoptotic cells. Alternatively, the cells can be stained with DAPI and analysed by flow cytometry. CellDeath.de - [Read DNA laddering assay for treated cells]

DNA-Methyltransferase Assay Protocol - http://www.mdanderson.org/departments/methylation/display.cfm?id=9B5B6082-C53A-4A86-BEE3EE149A52251E&method=displayFull&pn=A3F15D82-C9B4-41CD-BA4BE767E50A73D2

Category: Nucleic Acid Modification Protocols: DNA Methylation Protocols

Protocol for DNA-methyltransferase assay. Includes: Homogenize Cells/Tissues; Quantitate Protein Concentration; Dilute Samples; The Assay Proper; Troubleshooting. - [Read DNA-Methyltransferase Assay Protocol]

Enzyme-Linked ImmunoSorbent Assay (ELISA) for NGF Protocol - http://crutcherlab.med.uc.edu/ELI.htm

Category: Immunology: Immunoassays

Protocol for ELISA assay for NGF. Includes: ABSORPTION OF THE POLYCLONAL AND PREIMMUNE SERUM; BLOCKING; SAMPLE PREPARATION; PREPARATION OF NGF STANDARDS; PROTEIN RECOVERY; DESIGNING THE PLATE; APPLYING THE STANDARDS AND SAMPLES; APPLYING THE MONOCLONAL; APPLYING SECONDARY ANTIBODY; APPLYING STREPTAVIDIN; CHROMAGEN DEVELOPMENT; READING THE PLATE. - [Read Enzyme-Linked ImmunoSorbent Assay (ELISA) for NGF Protocol]

Articles (1-8 of 8)

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.

Quick DNA Agarose Gel Electrophoresis Protocol.

A quick and simple protocol for the electrophoresis of DNA fragments using agarose gels.

Absorption Spectroscopy and Quantification of Filamentous Phage Protocol

Unlike spherical phage, such as T4 and λ, which have roughly equal weight ratios of protein to DNA, filamentous phage have about six times more protein than DNA; the protein therefore contributes substantially to the absorption spectrum.

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.

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.