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Troubleshooting Western Blot - http://www.molecularstation.com/protein/troubleshooting-western-blots/

Category: Protein Protocols: Western Blot Protocol: Troubleshooting Western Blots

Covers Many Western Blot Problems and Includes Many Solutions. Fuzzy Bands, Low or Weak Signal, High Background, Spots on Film, Too Many Bands. A MolecularStation Guide. - [Read Troubleshooting Western Blot]

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Determination of the CpG Island Methylator Phenotype (CIMP) in colorectal cancer using MethyLight - http://www.natureprotocols.com/2006/06/30/determination_of_the_cpg_islan.php

Category: PCR Protocols: Real-Time PCR Protocols

Protocol provides a detailed, step-by-step protocol of the MethyLight assay for detection of CIMP with high sensitivity and specificity in colorectal cancer using a five marker panel composed of CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1. - [Read Determination of the CpG Island Methylator Phenotype (CIMP) in colorectal cancer using MethyLight]

Direct Selection of cDNAs with Large Genomic DNA Clones Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4077

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The goal of this method is to identify transcriptionally active genes in cloned segments of genomic DNA. The protocol uses hybridization and affinity purification to recover biotin-labeled cDNAs that bind to a 500-kb segment of human DNA cloned in a BAC vector. However, the method can be easily adapted to other clones of genomic DNAs cloned in high-capacity vectors. - [Read Direct Selection of cDNAs with Large Genomic DNA Clones Protocol]

DNA Electroelution - http://rothlab.ucdavis.edu/protocols/dna-electroelution.html

Category: DNA Protocols: DNA Extraction Protocols: DNA Extraction Polyacrylamide Gels

DNA Electroelution. This protocol describes the purification of DNA by trapping in a high-salt cushion in a "UEA AnalyticalElectroeluter" (IBI). This machine is no longer manufactured, to our knowledge. However, a smiliar device can be easily made from Plexiglas according to the following diagram, taken from Cornel Mulhardt, Molecular Biology and Genomics (2007) Academic Press, p.52: Schimenti Lab - [Read DNA Electroelution]

DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_124-126.pdf

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol describes here a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. Includes: In situ hybridization with DIG-labeled probes; Detection of DIG-labeled probes; Fluorescence microscopy. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System]

DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_124-126.pdf

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]

DNA Preparation and Needle Loading for Gene Delivery by Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4652

Category: Microinjection Protocols: Microinjection of DNA Protocols

For microinjection into cells, high-quality plasmid DNA is purified using standard procedures. The resulting preparation is then delivered into the microinjection needle by either a back-filling or a forward-filling approach. - [Read DNA Preparation and Needle Loading for Gene Delivery by Microinjection Protocol]

DNA Transfection Using Polybrene Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3875

Category: Transfection Protocols

Polybrene and DMSO can be used to achieve stable transformation of several types of cells by plasmid DNA. The yield of transformants is up to 15-fold greater with Polybrene than with calcium phosphate-DNA coprecipitation. However, there is no difference between the two methods in the efficiency of transformation of cells by high-molecular-weight DNA. - [Read DNA Transfection Using Polybrene Protocol]

Dnase-chip Protocol - http://www.natureprotocols.com/2006/07/03/dnasechip_a_high_resolution_me.php

Category: Microarray Protocols: Microarray Chip Protocols

Dnase-chip: A High Resolution Method to Identify DnaseI Hypersensitive Sites using Tiled Microarrays. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome. - [Read Dnase-chip Protocol]

DNaseI Footprinting Assay - http://www.fhcrc.org/science/labs/breeden/Methods/DNaseI-footprint.html

Category: DNA Protocols: EMSA DNA-Protein Protocols

DNaseI Footprinting Assay. Works well high affinity dna binding proteins. Breeden Lab. - [Read DNaseI Footprinting Assay]

E.coli Total RNA Labeling Protocol for High Density Oligonucleotide Array - http://www.genome.wisc.edu/resources/protocols/rnalablelingoligo.htm

Category: E Coli Protocols: E coli Nucleic Acid Protocols

E.coli total RNA labeling protocol for high density oligonucleotide array. Includes: RNA Preparation; Digest RNA and Purification of cDNA; Purify cDNA with Qiaquick PCR purification kit; cDNA Fragmentation and end labeling; Labeling with Terminal Transferase. - [Read E.coli Total RNA Labeling Protocol for High Density Oligonucleotide Array]

E.coli Total RNA Labeling Protocol for High Density Oligonucleotide Array - http://www.genome.wisc.edu/resources/protocols/rnalablelingoligo.htm

Category: Microarray Protocols: Microarray DNA Labeling Protocols

Protocol for e.coli total RNA labeling for high density oligonucleotide array. - [Read E.coli Total RNA Labeling Protocol for High Density Oligonucleotide Array]

ELISA Method Measure Inhibition COX Enzymes - http://www.natureprotocols.com/2006/11/22/an_elisa_method_to_measure_inh.php

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. - [Read ELISA Method Measure Inhibition COX Enzymes]

EMS Mutagenesis of Arabidopsis Seed Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/28/pdb.prot4621

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

EMS is used at concentrations that induce multiple point mutations in each plant, such that mutant alleles of a specific locus are found at a rate of ~1 in 2000-5000 M2 plants. This high rate of mutagenesis makes possible the screening of relatively few plants to find those with the phenotype of interest, a particular advantage if the screen is laborious or if only a small number of genes mutate to the required phenotype. - [Read EMS Mutagenesis of Arabidopsis Seed Protocol]

Experimental Procedures for Protein Microarrays - http://web.archive.org/web/20050313184601/http://cgr.harvard.edu/macbeath/protocols/proteinmicroarrays.html

Category: Protein Microarray Protocols

Experimental Procedures for Protein Microarrays. MacBeath G & Schreiber SL (2000). Printing proteins as microarrays for high-throughput function determination. Science 289: 1760-1763. - [Read Experimental Procedures for Protein Microarrays]

FPLC Protocol - http://users.path.ox.ac.uk/~ciu/Methods/Protein/fplc.html

Category: Chromatography Protocols

FPLC Protocol. The FPLC consists of a pump and a column which will withstand high pressure so separations can be carried out relatively quickly. For a detailed description there is a FPLC system handbook which is particularly useful for trouble shooting. For use of individual columns follow the "instructions" (in the green folder) which accompany each one. Sir William Dunn School of Pathology, Oxford University. - [Read FPLC Protocol]

Fractionation of Maize Embryo Proteins for 2-D Gel Electrophoresis Using Multicompartment Electrolyz - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4235

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

Protocol describes a method for performing isoelectric fractionation of a maize embryo sample using a multicompartment electrolyzer(MCE). This prefractionation of proteins having pIs within a certain pH interval is essential for allowing high loads of protein to be resolved on narrow and ultra-narrow immobilized pH gradients used in 2D electrophoresis. The isoelectric membranes in the MCE act like isoelectric traps capturing all the protein species having pIs encompassing the pI value of each... - [Read Fractionation of Maize Embryo Proteins for 2-D Gel Electrophoresis Using Multicompartment Electrolyz]

Fractionation of T and B Cells Using Magnetic Beads Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E66367B022BC1A53B67DF9C702B2513&objectid=66749A4CD26365F08DFBDE734F6A50F5

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

Protocol describes the purification of mouse T cells, B cells, and T cell subsets using magnetic bead separation. Isolation of cell subsets using magnetic beads is quick, simple, and reliable and can result in high yields of very pure cells. - [Read Fractionation of T and B Cells Using Magnetic Beads Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

G1 Synchronization of CHO Cells by Isoleucine Deprivation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4486

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: G Phase Protocols

This protocol provides a method for the synchronization of a monolayer culture of CHO cells in G1 using isoleucine deprivation. Since CHO cells can also be adapted to grow in suspension culture, this procedure can be used to obtain larger quantities of cells. When isoleucine is replaced, the cells resume growth and begin to enter S phase ~4 hours later. This method arrests almost 100% of the CHO cells in G1, and upon reversal, leads to rapid recovery of cell growth and very high cell viability. - [Read G1 Synchronization of CHO Cells by Isoleucine Deprivation Protocol]

G1 Synchronization of CHO Cells by Isoleucine Deprivation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4486

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

Genome-Wide Analysis Protocol - http://www.natureprotocols.com/2006/06/29/genomewide_analysis_of_data_ge.php

Category: Microarray Protocols: Microarray Analysis Protocols

Genome-wide analysis of data generated on the Affymetrix 10K Xba 142 arrays for identification of regions with high probability to contain genes responsible for Micronodular (non-pigmented) Adrenocortical Hyperplasia. - [Read Genome-Wide Analysis Protocol]

Growing Adherent Cells on Coverslips or Multiwell Slides Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4289

Category: Cell Culture Protocols

Glass is an excellent substrate for most tissue-culture-adapted cells and is compatible with all fixing and staining solutions. Glass coverslips in tissue-culture dishes or in 24-well multiwell plates are suitable carriers, as are multiwell slides. For high-resolution studies, choose glass coverslips of the highest available grade; #1 or #1.5 coverslips are the appropriate thickness. - [Read Growing Adherent Cells on Coverslips or Multiwell Slides Protocol]

Growth and Concentration of Chlorogonium for Culturing Freshwater Hypotrichs Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4464

Category: Laboratory Model Organisms Protocols

Protocol describes the growth and concentration of the alga Chlorogonium elongatum as a food source for culturing freshwater hypotrichs. Most freshwater hypotrichs (including Oxytricha nova, O. fallax, and O. trifallax; Euplotes aediculatus and E. eurystomous; and Stylonychia lemnae) can be grown to high density with Chlorogonium as the food organism. A similar regimen can be used to prepare other food sources such as Tetrahymena or bacteria (e.g., Aerobacter aerogenes). - [Read Growth and Concentration of Chlorogonium for Culturing Freshwater Hypotrichs Protocol]

Growth of S. cerevisiae and Preparation of DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3298

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes methods for isolation of DNA from a strain of S. cerevisiae carrying a recombinant YAC. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. The method is suitable for preparing DNA that will be used for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that do not require intact high-molecular-weight DNA. - [Read Growth of S. cerevisiae and Preparation of DNA Protocol]

High Efficiency FCC Preparation and Tx Protocol - http://genetics.med.harvard.edu/~cepko/protocol/mike/F4.html

Category: Competent Cells Protocols

This is an easy and straightforward protocol that gives efficiencies of 106 to 107 cfu/mg of plasmid DNA. A growth curve is required for each strain that is prepared.
- [Read High Efficiency FCC Preparation and Tx Protocol]

Articles (1-7 of 7)

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.

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.

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.

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.

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.

Vector Design Protocol for Transgenic Mice Generation

The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.

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