added Protocols

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for bidirectional, blunt-end cloning of DNA fragments.  The target DNA is PCR amplified and 3'-extensions are polished with Pfu DNA polymerase.  The amplicon is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent Escherichia coli.  A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Bidirectional Cloning of PCR Products Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

Once tissues are fixed and permeabilized, the antibodies are added. These antibodies can be labeled directly or detected by a labeled secondary reagent. For indirect detection, any reagent that binds specifically to the primary antibody can be "tagged" and used to locate the antibody. The possible reagents include anti-immunoglobulin antibodies, protein A or G, or, if the first antibody is labeled with biotin, streptavidin. They can be labeled with enzymes or gold. - [Read Binding Antibodies to Tissue Sections Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

This method, for the selective amplification of full-length cDNA ends, involves the addition of an adapter during reverse transcription.  This method takes advantage of the propensity of Moloney murine leukemia virus reverse transcriptase (MMLV RT) to append two to four cytosines to the 3'-end of newly synthesized cDNA strands.  The additional residues are added when the enzyme reaches the 5'-cap structure at the end of the mRNA template. - [Read Cap-Switching RACE Protocol]

Category: PCR Protocols: PCR Cloning Protocols

This method of direct cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable polymerases. - [Read Cloning PCR Products into T Vectors Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for Competitive RT-PCR.For quantifying mRNA, we use a competitive RT-PCR protocol with internal standard RNAs. These are added in a defined quantity to the RNA sample prior to the RT reaction. The resulting standard cDNA is coamplified with the s - [Read Competitive RT-PCR Protocol]

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]

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for directional blunt-end cloning of DNA fragments.  The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA.  The products of the ligation reaction are used to transform competent Escherichia coli.  A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Directional Cloning of PCR Products Protocol]

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

This protocol uses the plant amino acid mimosine as a G1/S synchronizing agent. Cells are first treated with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. Thymidine is then removed to allow all the cells to proceed completely through the S phase. Mimosine is then added to arrest the cells at the G1/S border. When mimosine is removed, cells will begin to enter S phase within about 1 hour. - [Read G1/S Phase Synchronization Using Mimosine Arrest Protocol]

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

Protocol uses the plant amino acid mimosine as a G1/S synchronizing agent. Cells are first treated with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. Thymidine is then removed to allow all the cells to proceed completely through the S phase. Mimosine is then added to arrest the cells at the G1/S border. When mimosine is removed, cells will begin to enter S phase within about 1 hour. - [Read G1/S Phase Synchronization Using Mimosine Arrest Protocol]

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

This protocol uses the plant amino acid mimosine as a G1/S synchronizing agent. Cells are first treated with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. Thymidine is then removed to allow all the cells to proceed completely through the S phase. Mimosine is then added to arrest the cells at the G1/S border. When mimosine is removed, cells will begin to enter S phase within about 1 hour. - [Read G1/S Phase Synchronization Using Mimosine Arrest Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. - [Read Immunoprecipitation: Preclearing the Lysate Protocol]

Category: Protein Protocols: Lowry Protein Assay

Lowry Protein Assay. The Lowry procedure is one of the most venerable and widely-used protein assays, being first described in 1951 [Lowry et al., J. Biol. Chem. 193: 265-275 (1951)]. Under alkaline conditions, copper complexes with protein. When folin phenol reagent (phospho-molybdic-phosphotungstic reagent) is added, the Folin-phenol reagent binds to the protein. Bound reagent is slowly reduced and changes color from yellow to blue. P.J. Hansen, Dept. of Animal Sciences, University of Florida. - [Read Lowry Protein Assay]

Category: Microinjection Protocols: Microinjection Equipment

This protocol contains methods for pulling microinjection needles using two different models of pipette pullers. The advantage of pulling needles in the laboratory is that a variety of different needle types can be pulled, depending on the samples and cells being injected. An added advantage is cost; once a pipette puller has been purchased, boxes of glass capillaries are inexpensive compared to premade microinjection needles. - [Read Preparation (Pulling) of Needles for Gene Delivery by Microinjection Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for preparation of messenger-dependent rabbit reticulocyte lysate. Informamtion also added to test that the lysate is working before doing the Nuclease inactivation - [Read Preparation of Messenger-Dependent Rabbit Reticulocyte Lysate]

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]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Solutions containing plasmid DNA are adjusted to a density of 1.55 g/ml with solid CsCl.  The intercalating dye, ethidium bromide, which binds differentially to closed circular and linear DNAs, is then added to a concentration of 200 mu;g/ml.  During centrifugation to equilibrium, the closed circular DNA and linear DNAs form bands at different densities. - [Read Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients]

Category: PCR Protocols: Quantitative PCR Protocols

Quantitative PCR involves co-amplification of two templates: a constant amount of a preparation containing the desired target sequence and serial dilutions of a reference template that is added in known amounts to a series of amplification reactions.  The concentration of the target sequence is determined by simple interpolation into a standard curve. - [Read Quantitative PCR Protocol II]

Category: Cell Biology and Cell Culture

Protocol for the isolation of the lipid-rich microdomains of the plasma membrane, notably caveolae and lipid rafts. Methods for the isolation of lipid rafts are based on the insolubility of these structures in the nonionic detergent TritonX-100. Either the intact cells are treated with a detergent-containing solution or a post-nuclear supernatant is prepared from a cell homogenate and then Triton X-100 is added to this supernatant. - [Read S20 Purification of detergent-insoluble lipid rafts from cells and tissues.]

Category: Mouse Protocols: Mouse Cell Culture Protocols

The liver of a rat is cannulated and perfused in situ with buffer, following which it is excised and perfused in a closed system with a collagenase solution. After a period of time the liver begins to break up, at which point it is transferred to a measuring cylinder and culture medium is added. It is then gently agitated to cause the release of cells which are subsequently filtered and allowed to settle out. The parenchymal and non-parenchymal cells form two distinct layers which can be separat - [Read The Isolation and Culture of Rat Hepatic Cells Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

When an individual bacterial virus grows in a bacterial host suspended in a top agar lawn, its progeny infect and lyse the surrounding host cells. This causes the appearance of a "hole" or plaque in the otherwise homogeneous bacterial lawn. Since each plaque represents a single virus, the number of viruses in the aliquot added to the plate is equal to the number of plaques which appear. - [Read Titering of Bacterial Viruses Protocol]

Category: Epigenetics and Methylation Protocols: 5-Aza-dC Treatment AZA Procedure

"Cells were seeded at a density of 1X106 cells/100-mm dish with 10% FBS and allowed to attach over a 24-h period. 5-Aza-dC (Sigma) was then added to a final concentration of 0.2–1
M, and the cells were allowed to grow for 5 days. The medium with or w - [Read Treatment of Cells with 5-aza-dC. protocol PDF]