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Blunt-end Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3830

Category: PCR Protocols: PCR Cloning Protocols

Protocol for blunt-end cloning of PCR products. Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves. I - [Read Blunt-end Cloning of PCR Products Protocol]

Cell Staining for Immunofluorescence Microscopy - http://www.bdbiosciences.com/pharmingen/protocols/Immunofluorescence_Microscopy.shtml

Category: Molecular Imaging: Immunofluorescence Microscopy

Cell Staining for Immunofluorescence Microscopy. Includes protocols for fixing the cells, Coverslip Preparation for Adherent Cells, Coverslip Preparation for Non-Adherent Cells, Paraformaldehyde Fixation, and Methanol/Acetone Fixation. Blocking protocols include blocking with primary antibody, and incubation with secondary antibody. - [Read Cell Staining for Immunofluorescence Microscopy]

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]

Detection FISH Protocol - http://www.riedlab.nci.nih.gov/publications/FISH%20Detection.pdf

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for Detection (FISH). TRITC, or Avidin Cy-5. For the probes labeled with digoxigenin, we usually first incubate with mouse-anti-digoxigenin, followed by incubation with sheep anti-mouse Cy5.5, or other fluorochrome conjugated antibodies. - [Read Detection FISH Protocol]

Factors that Influence Restriction Enzyme Digestion Activity - http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Factors that Influence Restriction Enzyme Digestion Activity. Buffer Composition, Incubation Temperature, DNA Methylation, Star Activity, Multiple Enzymes. Laura Austgen PhD et. al, Biomedical Hypertextbook, Colorado State Univ. - [Read Factors that Influence Restriction Enzyme Digestion Activity]

How to Avoid Pitfalls and Keep Out of Trouble in the Neurospora Lab - http://www.fgsc.net/neurosporaprotocols/How%20to%20avoid%20pitfalls%20and%20keep%20out%20of%20trouble.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to avoid pitfalls and keep out of trouble in the Neurospora lab. Includes: Culture, incubation, storage, and disposal; Stock preservation; Spore and colony counts; Making crosses and recovering progeny; Use of heterokaryons for allelism tests; Use of unordered asci to estimate the relation of translocation breakpoints to centromeres. - [Read How to Avoid Pitfalls and Keep Out of Trouble in the Neurospora Lab]

Immunocytochemistry - http://cancer.ucsd.edu/howelllab/Immunocyto.html

Category: Immunology: Immunocytochemistry Protocols

Method for plating cells, fixing cells, antibody incubation and mounting of slides. PHEM buffer, Antifade recipes. Howell Lab. - [Read Immunocytochemistry]

Immunohistochemistry with AP-Conjugated (NBT/BCIP) Protocol - http://icg.cpmc.columbia.edu/cattoretti/Protocol/immunohistochemistry/NBTBCIPstaining.html

Category: Immunohistochemistry Protocols

Protocol for immunohistochemistry with AP-Conjugated (NBT/BCIP). Protocol extensively blocks slides, further diluting the primary antibody, lengthening the incubation and washing time, using a simple AP-conjugated secondary at high dilution and use a slow long development with the most powerful IHC development, NBT/BCIP. Includes: Single AP stainiing and Double AP staining. - [Read Immunohistochemistry with AP-Conjugated (NBT/BCIP) Protocol]

Kit for Rapid Enrichment of SARS Virus - http://www.gentaur.com/sars_isolation.htm

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

During incubation period and the course of disease, SARS virus replicates and releases from cells. The level of virus varies greatly from sample to sample. At early stage of incubation period or during the late convalescent phase, the concentration of virus is very low in all kinds of samples. Virus is also detected at extremely low concentrations in plasma during... - [Read Kit for Rapid Enrichment of SARS Virus]

Long-Term Storage of Unfrozen Tetrahymena Cultures in Soybean Medium Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4472

Category: Tetrahymena Protozoa Protocols

Long-term storage of unfrozen Tetrahymena is convenient when a laboratory is not using the cells frequently; however, cells maintained under these conditions often take a couple of days of incubation in culture medium to recover. - [Read Long-Term Storage of Unfrozen Tetrahymena Cultures in Soybean Medium Protocol]

Metabolic Labeling & Immunoprecipitation Protocols - http://www.cbrinstitute.org/labs/springer/protocols/jun_35Slabeling_IP.html

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Cells incorporate 35S-methionine or cysteine during the protein synthesis. Thus it is essential to use Met,Cys-free medium and dialyzed FCS during the labeling. Short period of incubation with 35S-methionine or cysteine will result in radiolabeling (pulse), and additional incubation with excess concentration of unlabeled Met+Cys (chase) is needed for complex glycoproteins like integrins to get expressed as a maturated form. - [Read Metabolic Labeling & Immunoprecipitation Protocols]

Quantitative GUS Activity Assay in Intact Plant Tissue Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4688

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

The activity of ß-glucuronidase (GUS) can be accurately determined in intact plant tissue using 4-methylumbelliferyl ß-D-glucuronide (4-MUG) as a substrate. Upon hydrolysis by GUS, the fluorochrome 4-methyl umbelliferone (4-MU) is produced. This method is based on the permeability of both 4-MUG and 4-MU through plant tissue. It consists of incubation of the tissue with the reagent and quantification of the fluorescence emitted by 4-MU in the solution. GUS activity in each sample can be... - [Read Quantitative GUS Activity Assay in Intact Plant Tissue Protocol]

Removal of Cross-reactive Antibodies from Antiserum: Incubation with E. coli Lysate - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3767

Category: Antibody Protocols: Antibody Production Protocols

This protocol describes how antibodies that react with bacterial-encoded proteins may be removed from polyclonal antisera by incubation with a bacterial lysate. - [Read Removal of Cross-reactive Antibodies from Antiserum: Incubation with E. coli Lysate]

Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3772

Category: Antibody Protocols: Antibody Production Protocols

Antibodies directed against bacterial and phage-encoded proteins can be removed from sera by incubation with nitrocellulose filters impregnated with lysates of uninfected or phage-infected E. coli. - [Read Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening Protocol]

RNA Whole Mount In Situ Hybridization Protocol - http://genetics.med.harvard.edu/~cepko/protocol/ctlab/ish.ct.htm

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for RNA whole mount in situ hybridization. Includes: Embryo preparation; Prehybridization and Hybridization; Post-hybridization washes, blocking, and antibody incubation; Post-antibody washes; Color development. - [Read RNA Whole Mount In Situ Hybridization Protocol]

Transfection of Hippocampal Neurons with Plasmid DNA Using Calcium Phosphate Coprecipitation - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4445

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol describes transfection of plasmid DNA into primary hippocampal neurons using DNA/calcium-phosphate (CaPO4) coprecipitation. The precise pH of the transfection medium and the incubation time of cells with the coprecipitate are critical for reproducible and efficient transfection. Once these parameters are optimized for a given plasmid, the method is easily adapted for transfection of other established cell lines. - [Read Transfection of Hippocampal Neurons with Plasmid DNA Using Calcium Phosphate Coprecipitation]

Unmasking Hidden Epitopes with Proteases Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4528

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

Fixation can mask epitopes. However, it is often possible to re-expose them using a gentle incubation with proteases, which removes obstructing structures and allows antibody access, as described here. Many proteases can be used for this procedure, including very crude preparations of proteases, such as pronase. However, using a better-characterized protease, such as trypsin, allows a more controlled reaction and better comparison between experiments. - [Read Unmasking Hidden Epitopes with Proteases Protocol]

Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4506

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method for staining nerve fibers in whole-mount preparations of avian embryos using an antibody against the 160-kD subunit of neurofilaments. This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos. This protocol has been successfully applied for embryos at different stages up to about stage 33 (7 days of incubation). - [Read Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos]

Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4506

Category: Avian Bird Protocols

Articles (1-6 of 6)

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.

Antibodies Phage Expression Protocol Using 96-well Microtiter Plates

A Phage display expression protocol for antibody expression in 96-well microtiter plates.

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.

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.