obtained Protocols

Category: Bioinformatics

Using AFPredictor, it was demonstrated that ‘ordered surface carbons’ (OSCs) are a distinguishing feature of AFPs and, more specifically, their ice-binding surfaces. AFPredictor identified AFPs from within a large set of structures with greater than 99% specificity. Furthermore, it was used to identify a novel ice-binding protein by screening a library of homology modeled structures based on cDNA sequences obtained from cold-acclimated winter rye (Secale cereale). - [Read A Computational Screening protocol for Antifreeze/Ice-Structuring Proteins]

Category: Cytogenetics Protocols

Protocol for Agrobacterium-mediated transformation in rice. Agrobacterium-mediated rice transformation method that is applicable to easily cultured varieties in addition to elite japonica varieties that are more difficult to culture. Using this method, transgenic rice plants can be obtained in about 2–3 months with a transformation frequency of 30–50%, both in easily cultured varieties and recalcitrant elite japonica rice. - [Read Agrobacterium-Mediated Transformation in Rice Protocol]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

An Integrative Procedure for Apoptosis Identification and Measurement. Yingyu Cui Lab/Group: National Key Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences. I have uploaded an integrative procedure through which a relatively satisfactory result can be obtained following a single stage of cell culture and transient cell treatment, then detection with different instruments. This shortens experiment time. - [Read An Integrative Procedure For Apoptosis Identification And Measurement]

Category: DNA Protocols: DNA Extraction Protocols: Extracting Ancient DNA

The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, but PCR can be used to amplify and study short mitochondrial DNA sequences thatare of anthropological and evolutionary significance. SVANTE PAABO, PNAS. - [Read Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic amplification. PDF]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

An integrative procedure through which a relatively satisfactory result can be obtained following a single stage of cell culture and transient cell treatment, then detection with different instruments. This shortens experiment time. - [Read Apoptosis Identification and Measurement Protocol]

Category: DNA Protocols: DNA Extraction Protocols

Cheek cells obtained by rinsing the mouth commercial mouth wash solution. Mouth wash is then discarded into a sterile conical tube and sent to the lab. Based on Salting out procedure. DNA Laboratory, Medical School, Malta. - [Read DNA Extraction from Cheek Cells]

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: Immunology: Mononuclear Cell Isolation Protocols

Protocol for enrichment of PBMCs with monocytes. The cell suspension obtained after this protocol contains 40-70% monocytes. This cell suspension is than used for positive or negative MACS separation. - [Read Enrichment of PBMCs with Monocytes Protocol]

Category: Cloning Protocols

Protocol describes a method for DNA fragmentation by nebulization, in which the fine mist created by forcing a DNA solution through a small hole in the nebulizer unit is collected. The size of the fragments obtained by nebulization is determined chiefly by the speed at which the DNA solution passes through the hole, altering the pressure of the gas blowing through the nebulizer, the viscosity of the solution, and the temperature. - [Read Fragmentation of DNA by Nebulization Protocol]

Category: Microscopy Protocols: Electron Microscopy Protocols

In recent years, the increased sensitivity of electron detectors and the availability of low-vacuum or variable-pressure systems have allowed imaging of fresh tissue samples without the need for fixation, drying, and coating. This obviously saves a lot of time, although the image quality may not be as good as that obtained from fixed samples. However, for most applications that tend to be at a relatively low magnification, the quality can be as good as that obtained from fixed samples. - [Read Imaging of Fresh Arabidopsis Tissues in the Scanning Electron Microscope]

Category: Plant Biology Protocols

In recent years, the increased sensitivity of electron detectors and the availability of low-vacuum or variable-pressure systems have allowed imaging of fresh tissue samples without the need for fixation, drying, and coating. This obviously saves a lot of time, although the image quality may not be as good as that obtained from fixed samples. However, for most applications that tend to be at a relatively low magnification, the quality can be as good as that obtained from fixed samples. - [Read Imaging of Fresh Arabidopsis Tissues in the Scanning Electron Microscope Protocol]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates. Includes: Pretreatment of metaphase spreads on slides; Denaturation and hybridization; Single color fluorescent detection with immunological amplification; Multicolor fluorescence in situ hybridization (Multicolor FISH); Results obtained with human metaphase chromosome spreads. - [Read In Situ Hybridization to Human Metaphase Chromosomes using DIG-, Biotin- or Fluorochrome-Labeled DNA]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for the isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. - [Read Isolation of Arabidopsis Nuclei and Measurement of Gene Transcription Rates Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Method of choice when large amounts of mammalian DNA are required, for example, for Southern blotting (Rapid Isolation of Mammalian DNA, Rapid Isolation of Yeast DNA, Southern Blotting: Capillary Transfer of DNA to Membranes) or for construction of genomic libraries in bacteriophage {lambda} vectors.  Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 x 107 cultured aneuploid mammalian cells (e.g., HeLa cells). - [Read Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol]

Category: C. elegans Protocols

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. - [Read Live Imaging of Caenorhabditis elegans: Examples]

Category: Cytogenetics Protocols

Protocol describes a recently developed method — methylation-specific digital karyotyping (MSDK) — that enables comprehensive and unbiased genome-wide DNA methylation analysis. Using a combination of a methylation-sensitive mapping enzyme (for example, AscI) and a fragmenting enzyme (for example, NlaIII), short sequence tags can be obtained and uniquely mapped to genome location. - [Read Methylation-Specific Digital Karyotyping Protocol]

Category: Microinjection Protocols: Microinjection of Proteins Protocols

This protocol describes a method for microinjecting proteins into the nucleus or cytoplasm of adherent cells. Microinjection equipment can be obtained from a number of suppliers; this protocol has been used with the Narishige IM-200 air pressure regulator and the Leitz micromanipulator. Using this system, it is possible to microinject a constant volume within a 50% difference among cells. - [Read Microinjection of Protein Samples Protocol]

Category: RNA Protocols: cDNA Libraries Library

"useful screen phage library non-radioactive, and not need too many clones. Up to 5 clones can be obtained usually within one week." Methods.info - [Read PCR based Lambda λDNA library screening method]

Category: Mouse Protocols: Mouse Genetics Protocols

Protocol for the PCR of blood, hair or small tissue samples of mouse. Includes: Preparation of blood or a tissue sample obtained by ear punch for PCR; Preparation of hair shafts for PCR. - [Read PCR of Blood, Hair or Small Tissue Samples of Mouse Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

A. thaliana has a very small haploid genome and this makes obtaining DNA somewhat difficult. The most notable problem is that DNA is usually contaminated with polysaccharide which inhibit restriction enzymes as well as other DNA modifying enzymes. This problem is most easily solved by using young plants which have not accumulated as much polysaccharide as older plants. The best results are obtained with plants that are two to three weeks post germinated. - [Read Plant DNA Extraction Protocol]

Category: Immunology: T Cell Protocols

T cell hybridomas can be obtained by fusing activated T cells with tumor cells. A heterogeneous population of hybridomas can be cloned by limiting dilution to obtain hybridomas that express specificity to one T cell receptor (TCR). Protocol describes cell fusion and selection of T cell hybridomas. A protocol is provided for screening of T cell hybridomas for expression of the CD3-TCR complex by flow cytometry analysis. - [Read Production of Mouse T Cell Hybridomas Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. Chromatin dynamics can be followed easily, and wild-type embryonic cells can be compared with mutants or RNAi-treated embryos. - [Read Protocol Live Imaging of Caenorhabditis Elegans]

Category: DNA Protocols: DNA Extraction Protocols: Agarose Gel DNA Extraction Protocols

Rapid Elution of DNA Agarose Gels Protocol.  This method allows quick purification of DNA fragments from agarose gels for use in cloning and other reactions. Higher yields of purified DNA can be obtained from commercially available purification kits, however greater ligation efficiencies per given amount of DNA have been seen with the use of these described spin columns. Hahn Lab. - [Read Rapid Elution of DNA Agarose Gels Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Protocol to be used as reference to help standardization of both the isolation procedure and the quality of the cultures allowing a better comparison of the results obtained from different laboratories. - [Read Rat Chromaffin Cells Primary Cultures: Standardization and Quality Assessment for Single-Cell Assay]

Category: Fungi Protocols: Neurospora Protocols

DNA isolation method yields an average of 0.6 micrograms of genomic DNA that is suitable for Southern analysis or PCR. Starting with fresh mycelium, 20 to 40 samples can be processed in approximately two hours. Better yields (about 5 micrograms) may be obtained by suspending approximately 100 microliters of ground lyophilized mycelium in 500 microliters of isolation buffer and following the protocol. - [Read Small Scale DNA Preps for Neurospora crassa Protocol]