amount Protocols

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

The protocol includes: organelle isolation, deoxyribonuclease treatment, lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple samples of organelle DNA from a small amount of tissue. - [Read A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower]

Category: PCR Protocols: Bisulfite Conversion Based PCR Protocols

Protocol for the analysis of DNA methylation using bisulphite sequencing. Method allows precise analysis of methylation in a certain region by converting all nonmethylated cytosines into tymines, while methylated cytosines remain unchanged.  This method requires small amount of genomic DNA and therefore seems to be very useful for the analysis of clinical samples, where the material amount is limited.
- [Read Analysis of DNA Methylation using Bisulphite Sequencing Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Different cell types vary in their phosphatidylserine (PS) content, along with the amount of PS exposure on the cell surface after cell death. This protocol is a guideline for getting started, however it may be necessary to adjust the concentration of the Annexin V-FITC. - [Read Annexin V Protocol for Flow Cytometry]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Method describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts]

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

Protocol describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

The recommended amount of RSV-ß-Galactosidase plasmid to use for transfection of cells (60 mm or 100 mm dish) is 1-2 µg. The optimal amount of plasmid DNA will be determined by the efficiency of transfection , which is very dependent upon the particular cell line and transfection protocol. - [Read B- Galactosidase Assay Protocol]

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]

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

A detailed and well thought out Bradford protein assay page using a spectrophotometer. Includes information such as the Bradford assay is very fast and uses about the same amount of protein as the Lowry assay, comments, procedural steps, equipment used and more. - [Read Bradford Protein Assay]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This calcium phosphate transfection method works best in cell lines that are 1) highly transformed and 2) adherent (Hela, U2OS, SAOS2, AdAH, NPC-KT and obtain from 20% to 100% transfection efficiency depending on the cell line). Works well for transient experiments but precautions should be used in the design and interpretation of experiments based on the discussion below. Also works very well for generating stable cell lines. This method is quite sensitive to the amount of input plasmid. - [Read Calcium Phosphate Transfection Method]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture. Detection is based on using the luciferase reaction to measure the amount of ATP from viable cells. The amount of ATP in cells correlates with cell viability. - [Read Cell Viability Assays that Measure ATP Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In this protocol, sample and competitor RNAs are reverse transcribed (separately) in a pilot experiment.  A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. Procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor.  The experiment includes controls for sample-to-sample variations in RT efficiency. - [Read Competitive RT-PCR: Estimation of Copy Number Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

In this protocol, the DNA-binding capacity of Wizard MagneSil particles is used to capture and release a consistent amount of DNA (100 ng) across a wide range of samples.  At the end of the procedure, the DNA is eluted into 100 µl Elution Buffer to give a final concentration of 1 ng/µl, relieving the need for postpurification DNA quantitation. - [Read DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs Protocol]

Category: Transfection Protocols

Protocol should be viewed as a starting point for systematic optimization of transfection mediated by lipofecting agents. Once a positive signal has been obtained from a transfected plasmid carrying a standard reporter gene, optimal conditions for transfection can be established by systematic variation of parameters such as the initial cell density, the amount and purity of DNA, the media and serum, and the time of exposure of the cells to the cationic-lipid-DNA complex. - [Read DNA Transfection Mediated by Lipofection Protocol]

Category: Developmental Biology: Embryology Protocols

The FAM caspase binding assay kits from ATCC Corporation can be used to determine amounts of active caspases in cells. The FAM-labeled caspase inhibitor can freely diffuse into the cell. Active caspase irreversibly binds the inhibitor. Upon washing the cells, the amount of fluorescence is proportional to the amount of active caspase in the cell. FAM-LETD-fmk (catalog no. 30-1306) is used to detect caspase 8 and FAM-LEHD-fmk (catalog no. 30-1308) is used for caspase 9. - [Read Fam Caspase 8 and 9 Binding Assay for Embryos Protocol]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for constant-flow microinjection using the Pneumatic PicoPump (World Precision Instruments). This type of system is very simple and can be assembled on a relatively low budget. In this method, a constant flow of sample is delivered from the tip of the pipette, and the amount of sample injected into the cell is determined by how long the pipette remains in the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Controlled-Flow System Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

For immunoblotting experiments, it is often important to compare the total amount of an antigen from many different sources or to learn if a particular source has the antigen under study.  In the approach described here, tissue cultures, bacteria, yeast cells, tissues, and other sources of antigens are disrupted directly in an electrophoresis sample . - [Read Immunoblotting: Preparing Cell Lysates Protocol]

Category: Yeast Protocols

Protocol for in vitro endoplasmic reticulum to golgi transport reaction in Yeast. Includes: Preparation of Membranes; One-Stage Reaction; Two-Stage Reaction using Normal Amount of Membranes; Two-Stage Reaction using Low Concentration of Membranes. - [Read In Vitro Endoplasmic Reticulum to Golgi Transport Reaction in Yeast Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations (protocol may be found at www.methods.info). Reduction of the amount of microbeads in comparison to Miltenyi protocol reduces the costs of the experiment. - [Read Isolation of Monocytes from Enriched PBMCs using CD14 Magnetic Beads Protocol]

Category: Cell Biology and Cell Culture

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations. Reduction of the amount of microbeads reduces the costs of the experiment. - [Read Isolation of monocytes from monocyte enriched PBMC fraction using CD14]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Two methods are provided for purifying glycoproteins using wheat-germ agglutinin or concanavalin A-Sepharose.  Because lectin-affinity matrices can bind a few milligrams of protein per milliliter of affinity matrix, only a small amount of affinity gel matrix is required.  The batchwise method is recommended when protein volume is large. - [Read Lectin-Agarose Affinity Chromatography Protocol]

Category: Cloning Protocols

Protocol for ligating plasmid and target DNAs in low-melting-temperature agarose. Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. - [Read Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

5 ml liquid lysates are prepared when a small amount of DNA from a large number of lambda clones is needed. The lysates can be made using 10- 20 ul of a stock lysate or a 100-fold amplified phage "macroplaque" as the inoculum. - [Read Liquid Phage Lysates Protocol]

Category: Protein Protocols: Protein Quantitation Concentration Protocols

Find a list of assays for the determination of protein concentration in a solution.  This list includes the sensitivity range, volume/amount of sample needed, subjective comments on accuracy and convenience, and major interfering agents.  Procedural details, equipment requirements, and references are outlined in the individual assay documents. - [Read List of Protein Assays]

Category: Reporter Gene Assays: GFP Assay Protocols

Protocol measuring the amount of GFP expression and DNA content in permeabilized cells. Includes: Fix cells with formaldehyde; Permeabilize cells with ethanol; Preparation of Buffered 2% Formaldehyde Solution. - [Read Measurement of GFP Expression and DNA Content in Permeabilized Cells]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Standard PCR Protocol

PCR and Multiplex Guide. Amazing Guide to primer amount, MgCl2 amount, annealing temperature, and more. With picture examples. Octavian Henegariu. - [Read PCR and Multiplex Guide]