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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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A high yield affinity purification method for specific RNA-binding proteins. - http://www.pubmedcentral.gov/pagerender.fcgi?tool=pmcentrez&pageindex=1&artid=330202

Category: RNA Protocols: RNA-binding Protein Purification

A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta. Neupert 1990. - [Read A high yield affinity purification method for specific RNA-binding proteins.]

A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed - http://www.rnajournal.org/cgi/reprint/9/3/364.pdf

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

PDF. A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed in Pichia pastoris. CHARLES R. ALLERSON, ALAN MARTINEZ, EMINE YIKILMAZ and TRACEY A. ROUAULT. RNA Journal. - [Read A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed]

A Sealed Preparation for Long-Term Observations of Cultured Cells - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4660

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a sealed preparation that allows the continuous long-term observation of cultured mammalian cells on upright or inverted microscopes without environmental CO2 control. The preparation allows for optical conditions consistent with high-quality imaging and good cell viability for at least 100 hours. - [Read A Sealed Preparation for Long-Term Observations of Cultured Cells]

A Simplified System for Rapid Generation of Recombinant Adenoviruses - http://www.coloncancer.org/adeasy/protocol.htm

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

A simplified system for rapid generation of recombinant adenoviruses. Includes: Generation of Recombinants in Bacterial Cells; Viral Production in 293 or 911 Cells; Preparation of High Titer Viral Stocks. - [Read A Simplified System for Rapid Generation of Recombinant Adenoviruses]

Adaptation of High Five™ Cells to HyQ© SFX Insect Medium - http://www.hyclone.com/pdf/procedure_insect_highfive.pdf

Category: Cell Culture Protocols: Insect Cell Culture Protocols

This protocol is designed to adapt High Five™ (BTI-Tn-4) cells to serum-free suspension culture in HyQ SFX-Insect in 5 passages. The entire adaptation process takes approximately two weeks and the resulting adapted cells have doubling times of 16 and 20 hours and minimal clumping in suspension culture. - [Read Adaptation of High Five™ Cells to HyQ© SFX Insect Medium]

Amplification of Baculovirus Plaques and Preparation of High Titer Virus Stocks Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p608

Category: Cloning Protocols: Baculovirus Protein Expression Protocols

Protocol for amplification of Baculovirus plaques and preparation of high titer virus stocks. Includes: Primary Amplification; Secondary Amplification. - [Read Amplification of Baculovirus Plaques and Preparation of High Titer Virus Stocks Protocol]

Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4115

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR. A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems. The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization. - [Read Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium]

Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1 - http://www.cyto.purdue.edu/flowcyt/research/cytotech/apopto/data/cossar1/cossariz.htm

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]

Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography - http://www.glycotech.com/protocols/Proto6.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

High performance liquid affinity chromatography (HPLAC) is a useful procedure to investigate he interactions between carbohydrate binding protein and their ligands. Technical requirements are similar to conventional HPLC. HPLAC can screen and separate natural ligands from complex biological mixtures. WeiTong Wang~GlycoTech Corporation, Rockville, Maryland - [Read Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography]

Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography - http://www.glycotech.com/protocols/Proto6.html

Category: HPLC Protocols

Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography Protocol. Glycotech. - [Read Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography]

Antibiotics - http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/Materials/antibiotics.html

Category: Microbiology: Antibiotics Concentration in Media LB

Antibiotic stock solutions preparation, Storage conditions for antibiotics, Working Concentrations for high copy plasmids. Kay Schneitz Univ Zurich. - [Read Antibiotics]

Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4527

Category: Drosophila Protocols

Protocol for antibody addition to Drosophila specimens and detection using fluorochrome-linked reagents. Fluorochrome-linked reagents should be used when high resolution is needed or if two antigens need to be localized simultaneously. Because of the thickness of fly specimens, detection requires access to a confocal microscope. - [Read Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol]

Automated Imaging-Based High-Throughput Screening: Small Molecules that Reverse Cellular Senescence - http://www.natureprotocols.com/2006/06/30/automated_imagingbased_highthr.php

Category: Cell Biology and Cell Culture

Protocol for automated imaging-based high-throughput screening for small molecules that reverse cellular senescence. - [Read Automated Imaging-Based High-Throughput Screening: Small Molecules that Reverse Cellular Senescence]

Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3872

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol,is based on the venerable method of Graham and van der Eb (1973). The procedure is used chiefly to generate stable lines of cells carrying chromosomally integrated copies of the transfected DNA. - [Read Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA]

CAT.INIST.FR - http://cat.inist.fr/?aModele=afficheN&cpsidt=16477965

Category: Molecular Model Organisms: Mouse Animal Protocols: Mouse Genotyping Protocols

We have established a high-throughput protocol for mouse genotyping using two robotics workstations: a liquid-handling robot to assemble PCR and a ... - [Read CAT.INIST.FR]

Centrifuge Safety - High Speed Hazards - http://www.ehs.wsu.edu/Factsheets/FAQCentrifuge.asp

Category: General Research Protocols and Methods: Centrifuge Use and Centrifugation

Centrifuge Safety - High Speed Hazards. Safe Centrifuge Use , Care and Maintenance, and maintaining a log book for centrifuges. EHS Washington state Univ. - [Read Centrifuge Safety - High Speed Hazards]

Chambers for Examination of Live Cells under Mechanical Stress Protocol - http://www.cshprotocols.org/cgi/content/extract/2007/3/pdb.ip32

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Manual measurement and manipulation of the cell surface requires access to the cells, usually in an open chamber. Temperature-controlled chambers or stage inserts are preferred for maintaining physiological activity during the experiment. For example, heated culture dishes with coverslip glass bottoms (Bioptechs) permit high-resolution fluorescence microscopy of living cells during force application. - [Read Chambers for Examination of Live Cells under Mechanical Stress Protocol]

ChIP on ChIP Protocol PDF - http://genomecenter.ucdavis.edu/farnham/farnham/protocols/MOJO%20Dec%202003%20ChIP-chip%20protocol.pdf

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Method to identify genomic targets of DNA-binding factors. Chromatin immunoprecipitation (ChIP) assay on high-throughput microarray based methods for discovering genomic regions occupied by human DNA-binding proteins. Oberley and Farnham. - [Read ChIP on ChIP Protocol PDF]

Construction of Genomic DNA Libraries in Cosmid Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4000

Category: DNA Protocols: Cosmid Library Protocols

Protocol describes how to construct a library of 35-45-kb fragments of genomic DNA in the double cos site cosmid vector, SuperCos-1. The steps include: Linearization and dephosphorylation of SuperCos-1 DNA; Partial digestion of high-molecular-weight DNA with MboI; Dephosphorylation of high-molecular-weight genomic DNA; Ligation of cosmid arms to genomic DNA: Packaging and plating recombinants; Isolation and analysis of recombinant cosmids: Validation of the library. - [Read Construction of Genomic DNA Libraries in Cosmid Vectors Protocol]

Culture Chambers for Live-Cell Imaging - http://www.microscopyu.com/articles/livecellimaging/culturechambers.html

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Specimen chambers have had many designs published over the years describing systems that offer excellent optical properties while allowing specimens to be maintained for varying amounts of time. Ranging in complexity from the simple preparation of a sealed coverslip on a microscope slide to sophisticated perfusion chambers that enable tight control of virtually all environmental variables culture chambers are designed to to allow living specimens to be observed with minimal invasion at high res. - [Read Culture Chambers for Live-Cell Imaging]

Culturing of Insect Cells and Production of Baculovirus Expressed Recombinant Proteins in 2.8 Liter - http://www.hyclone.com/pdf/procedure_insect_fernbach.pdf

Category: Cell Culture Protocols: Insect Cell Culture Protocols

The use of Ferbach flasks is an ideal method to amplify the cells and virus inoculum for the bioreactor. The method is also very practical for producing sufficient amounts of various proteins for feasibility studies using HyQ© SFX-Insect™ medium. The large surface area in this vessel enables sufficient aeration and, therefore, the cells, when they reach high density, are not oxygen depleted. - [Read Culturing of Insect Cells and Production of Baculovirus Expressed Recombinant Proteins in 2.8 Liter]

Delivery of dsRNA into Drosophila Embryos by Gene Gun Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4517

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how subcellular-sized particles are accelerated to high velocity to carry double-stranded RNA (dsRNA) into Drosophila embryos. The major advantage of this procedure over microinjection (Microinjection of dsRNA into Drosophila Embryos) is that particle bombardment is easier and faster to perform. In addition, the mechanical trauma received is far less than by microinjection, allowing better survival of embryos and fewer phenotypic artifacts. - [Read Delivery of dsRNA into Drosophila Embryos by Gene Gun 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]

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: Genetics and Genomics: Cancer Genetics Protocols

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]

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: Nucleic Acid Modification Protocols: DNA Methylation Protocols

Determination of the CpG Island Methylator Phenotype (CIMP) in colorectal cancer using MethyLight. Protocol describes 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]

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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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