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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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Long-PCR Reagents and Guidelines - http://wheat.pw.usda.gov/~lazo/methods/lazo/longpcr.html

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Lysate Microarrays - Information - http://cgr.harvard.edu/macbeath/research/lysate_ma.html

Category: Protein Microarray Protocols

Lysate Microarrays. High throughput methods that detect abundance and post-translational modification. MacBeath Lab - [Read Lysate Microarrays - Information]

Method for Assaying Gliotoxin Production in Aspergillus fumigatus Protocol - http://www.aspergillus.org.uk/indexhome.htm?secure/laboratory_protocols/./gliotoxin1.html~main

Category: Fungi Protocols: Aspergillus Protocols

Gliotoxin is a metabolite of Aspergillus fumigatus that exhibits immunosuppressive activity against certain cells of the immune system. Secretion of gliotoxin during infection has been suggested as being a factor in the pathogenesis of aspergillosis. Gliotoxin secretion can be assayed in a number of ways by thin layer chromatography (TLC) high performance liquid chromatography (HPLC) or bioassay using the effect of gliotoxin on human cells1. - [Read Method for Assaying Gliotoxin Production in Aspergillus fumigatus Protocol]

Method: Preparation of Lymphocyte Cell Pellet for Storage - http://humgen.wustl.edu/hdk_lab_manual/hcc/hcc5.html

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Following propagation to 1 X 108 cells, lymphoblastoid cells are conveniently stored at -80 degrees C to preserve the high molecular weight DNA in the cells until the DNA is purified. This procedure describes the steps required to harvest and freeze the c - [Read Method: Preparation of Lymphocyte Cell Pellet for Storage]

Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorec - http://nar.oxfordjournals.org/cgi/content/full/33/5/e46

Category: DNA Microarray Protocols: DNA Methylation Microarray CpG

Kimura et al., NAR 2005. "easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylatio - [Read Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorec]

Microsome High Speed Pellet and Cytosol Preparation for Endoplasmic Reticulum to Golgi Transport - http://www.bio.com/protocolstools/protocol.jhtml?id=p1841

Category: Yeast Protocols

Protocol for microsome high speed pellet and cytosol preparation for endoplasmic reticulum to golgi transport assay in Yeast. - [Read Microsome High Speed Pellet and Cytosol Preparation for Endoplasmic Reticulum to Golgi Transport]

Microsome High Speed Pellet and Cytosol Preparation for Endoplasmic Reticulum to Golgi Transport Ass - http://www.bio.com/protocolstools/protocol.jhtml?id=p1841

Category: Cell Organelle Protocols: Microsome Protocols

Protcol relating to microsome high speed pellet and cytosol preparation for endoplasmic reticulum to golgi transport assay in yeast. Includes spheroplast formation. - [Read Microsome High Speed Pellet and Cytosol Preparation for Endoplasmic Reticulum to Golgi Transport Ass]

Microspheres for High-Throughput Screening Assays - http://www.pall.com/pdf/oep_hts_brochure.pdf

Category: Microsphere Analysis Protocols

The role of microspheres in these screens is similar to their traditional role in immunoassays, namely as a solid phase to either enhance detection, separation, or both. The predominance of radioactive assays in high-throughput screening, along with the desire to find alternative means of detection, have led to research on substituting alternative fluorescent technologies. - [Read Microspheres for High-Throughput Screening Assays]

Northern Hybridization Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3723

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for northern hybridization. Protocol describes how to carry out northern hybridization at high stringency in phosphate-SDS-buffers. Although a wide variety of formats are available, hybridization is usually performed in heat-sealable bags, roller bottles, or plastic boxes, as described here. - [Read Northern Hybridization Protocol]

Nuclear and Splicing Extract Preparation - http://penguin.uchc.edu/~intron/Protocols/NuclearS100%20Extract.html

Category: Protein Protocols: Nuclear Extraction Protocols

S100 extract cytosolic extraction preparation. Buffer A (Hypotonic Buffer), Low and High salt buffer recipes. Brent Graveley - [Read Nuclear and Splicing Extract Preparation]

Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4449

Category: Transfection Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. Cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. Regardless of cell size or phenotype, transfection efficiency increases with a high concentration of cells in a small volume. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Packing Ceramic Hydroxyapatite Columns for High-Performance Liquid Chromatography Protocol - http://cshprotocols.cshlp.org/cgi/content/extract/2006/9/pdb.prot4267

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol describes the packing of ceramic hydroxyapatite for HPLC. - [Read Packing Ceramic Hydroxyapatite Columns for High-Performance Liquid Chromatography Protocol]

PCR Program Design - http://info.med.yale.edu/genetics/ward/tavi/p08.html

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: PCR Optimization Protocols

The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. P - [Read PCR Program Design]

PCR Program Design - http://info.med.yale.edu/genetics/ward/tavi/p08.html

Category: PCR Protocols: PCR Optimization

Placenta Specific Gene Manipulation by Transducing Zona-Free Blastocyst using Lentiviral Vector - http://www.natureprotocols.com/2007/01/24/placenta_specific_gene_manipul.php

Category: Cytogenetics Protocols

Details a placenta specific gene manipulation by transducing blastocysts with lentiviral vectors1. After a removal of zona pellucida which functions as a physical barrier, trophoblast cells lying outermost layer of blastocyst were transduced from outside with high-titer lentiviral vectors. As most placental cells descend from trophoblast cells while fetus originated from inner cell mass, transgene expression can be observed in trophoblast cells from preimplantation stages and in placenta... - [Read Placenta Specific Gene Manipulation by Transducing Zona-Free Blastocyst using Lentiviral Vector]

Plasmid Transformation of Yeast Colonies Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4146

Category: Yeast Protocols: Yeast Transformation Protocols

Protocol for plasmid transformation of Yeast colonies. Protocol can be used if high efficiency is not needed. - [Read Plasmid Transformation of Yeast Colonies Protocol]

Precipitation of Bacteriophage {lambda} Particles from Large-scale Lysates Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3966

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Bacteriophage {lambda} particles are recovered from bacterial lysates by precipitation with polyethylene glycol at high ionic strength. - [Read Precipitation of Bacteriophage {lambda} Particles from Large-scale Lysates Protocol]

Preparation and High-efficiency Transformation of Competent E. coli - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3942

Category: Bacteria Transformation Protocols

Protocol generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/µg of superhelical plasmid DNA). - [Read Preparation and High-efficiency Transformation of Competent E. coli]

Preparation and Loading of Protein Samples for Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4656

Category: Microinjection Protocols: Microinjection of Proteins Protocols

his protocol provides methods for the preparation of protein samples and for loading them into pulled microinjection pipettes. Stock solutions of proteins are thawed, diluted (if desired), centrifuged at high speed to remove aggregates, and kept on ice until loading. Loading into micropipettes can be done using either a "front-loading" or a "backfilling" procedure. - [Read Preparation and Loading of Protein Samples for Microinjection Protocol]

Preparation and Transformation of Competent E. coli Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3942

Category: E Coli Protocols: E coli Transformation Protocols

Procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/µg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically. - [Read Preparation and Transformation of Competent E. coli Protocol]

Preparation of an Ion-Exchange Column Protocol - http://cshprotocols.cshlp.org/cgi/content/extract/2006/1/pdb.prot4195

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the preparation of ion-exchange chromatography column. Ion-exchange chromatography (IEC) can be used as a crude step in a protein purification scheme, or, with proper preparation, as a high-resolution step. If high resolution is desired, considerable care should be taken during column preparation, choice of IEC media, and column packing. - [Read Preparation of an Ion-Exchange Column Protocol]

Preparation of BAC DNA with CONCERT High Purity Plasmid Purification System Protocol - http://intramural.nimh.nih.gov/lcmr/snge/Protocols/BACProtocols/BACColumn.html

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol for the preparation of BAC (Bacterial Artificial Chromosome) DNA with CONCERT high purity plasmid purification system. Includes: - [Read Preparation of BAC DNA with CONCERT High Purity Plasmid Purification System Protocol]

Preparation of Electrolyte Gradient Gels Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3795

Category: Nucleic Acid Electrophoresis Protocols

Protocol for the preparation of electrolyte gradient gels. Electrolyte gradients are formed when buffers of different concentrations are used in the upper (low electrolyte concentration) and lower (high electrolyte concentration) chambers of the electrophoresis device. - [Read Preparation of Electrolyte Gradient Gels Protocol]

Preparation of High Titer Adenovirus in P11 Cells Protocol - http://www.whitelabs.org/Lab%20Protocols/adenovirus%20protocols/Preparation%20of%20High%20Titer%20Adenovirus%20in%20P11%20cells.htm

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol for the preparation of high titer adenovirus in P11 cells. - [Read Preparation of High Titer Adenovirus in P11 Cells Protocol]

Preparation of RNA from Plant Tissue Using Trizol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4105

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Protocol is based on the standard Trizol protocol for the purification of RNA from animal cells using Trizol (Purification of RNA from Animal Cells using Trizol). In this version, adapted for use with plant tissues, a high-salt isopropanol precipitation step has been added to precipitate RNA selectively, while maintaining polysaccharides and proteoglycans in solution. - [Read Preparation of RNA from Plant Tissue Using Trizol]

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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