roche Protocols

Category: Signalling Signal Transduction Protocols

Protocol for Akt/PKB kinase assay. Protocol includes: Akt lysis buffer; Kinase Buffer; 1.5x Akt kinase reaction buffer; Start solution; Lipofectamine PLUS; CDNA synthesis (Roche #1483?88). - [Read Akt/PKB Kinase Assay]

Category: Cell Biology and Cell Culture: Apoptosis Protocols

Apoptosis, Cell Death, And Proliferation. Roche Manual. Includes a complete list of apoptosis methods. Roche. - [Read Apoptosis, Cell Death, And Proliferation. Roche Manual.]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for Chem-Link labeling of DNA or RNA with DIG or Biotin. Includes: Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Choosing the right labeling method for your hybridization experiment. Includes: Homogeneous labeling methods for DNA; Homogeneous labeling methods for RNA; Stability of probe-target interaction; Nonradioactive labeling of oligonucleotides; Double-stranded versus single-stranded probes. - [Read Choosing the Right Labeling Method for your Hybridization Experiment]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. Includes: Cell preparations and BrdU labeling; Detection of antigen by immunocytochemistry (ICC); Visualization of ICC antigen; -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy); Cell processing for in situ hybridization; In situ hybridization (ISH); etc... - [Read Combined DNA In Situ Hybridization and Immunocytochemistry Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]

Category: Cytogenetics Protocols

CGH provides a comprehensive picture of all chromosomal gains and losses within a single experiment. In contrast to banding analysis, CGH requires no preparation of metaphase chromosomes from the cells to be analyzed (which in certain tumor types may be difficult or even impossible). - [Read Detection of Chromosomal Imbalances Using DOP-PCR and Comparative Genomic Hybridization (CGH)]

Category: Cytogenetics Protocols: Spectral Karyotyping SKY Protocols

Protocol for the Detection of chromosomal imbalances using DOP-PCR and comparative genomic hybridization (CGH). Includes: Preparation and labeling of DOP-PCR products; Comparative genomic hybridization (CGH). - [Read Detection of Chromosomal Imbalances using DOP-PCR and Comparative Genomic Hybridization (CGH)]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol for detection of even-skipped transcripts in Drosophila embryos with PCR/DIG-labeled DNA probes. - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of even-skipped transcripts in drosophila embryos with PCR/DIG-labeled DNA probes. This protocol has been used to detect the transcript distribution of a number of genes by in situ hybridization, including evenskipped and seven-up, in whole mount Drosophila embryos, and engrailed Antennapedia in whole mount grasshopper embryos. Includes: Probe labeling; Evaluation of labeling reaction; Preparation of embryos, hybridization and detection.
 - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of HPV 11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe. Includes: Probe preparation; Pretreatment of slides; Preparation of tissue sections; Hybridization; Washes; Detection of hybrids. - [Read Detection of HPV 11 DNA in Paraffin-Embedded Laryngeal Tissue with a DIG-Labeled DNA Probe Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes. - [Read Detection of mRNA in Tissue Sections Using DIG-labeled RNA and Oligonucleotide Probes Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes. - [Read Detection of mRNA on Paraffin Embedded Material of the Central Nervous System with DIG-Labeled RNA]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNAs on cryosections of the cardiovascular system using DIG-labeled RNA probes. Protocol was optimized from a protocol using 35S-labeled RNA probes. It allows to detect the expression of low abundant mRNAs in the cardiovascular system, e.g. of the proinflammatory cytokine GM-CSF in normal human coronary arteries, and of IL6 and gp130 in human failing hearts. The protocol can be combined with immunohistochemistry. - [Read Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol describes here a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. Includes: In situ hybridization with DIG-labeled probes; Detection of DIG-labeled probes; Fluorescence microscopy. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes.  The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization.  This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. 
    - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for estimating the yield of DIG-labeled nucleic acids. Includes: Estimating the yield in a spot test with a DIG-labeled control. - [Read Estimating the Yield of DIG-labeled Nucleic Acids Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Labeling Protocols

Protocol for estimating the yield of DIG-labeled nucleic acids. - [Read Estimating the Yield of DIG-Labeled Nucleic Acids Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension. Hybridization technique which does not need formamide and dextran sulfate. As a model system, we used the repetitive specific human DNA probe pUC 1.77, labeled it with digoxigenin-11-dUTP by nick-translation, and hybridized it to metaphase chromosomes in suspension. These chromosomes were isolated by standard techniques from human lymphocytes. - [Read Fluorescence In Situ Hybridization of a Repetitive DNA Probe to Human Chromosomes in Suspension]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for the identification of single bacterial cells using DIG-labeled oligonucleotides. Includes: Organisms and growth conditions; Cell fixation and preparation of cell smears; DIG labeling of oligonucleotides with DIG-ddUTP; In situ hybridization using digoxigenin-labeled oligonucleotides; Detection of DIG-labeled oligonucleotides with fluorescently labeled anti-DIG Fab fragments; Detection of DIG-labeled oligonucleotide.
 - [Read Identification of Single Bacterial Cells using DIG-Labeled Oligonucleotides Protocol]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for identification of single bacterial cells using DIG-labeled oligonucleotides. Includes: Organisms and growth conditions; Cell fixation and preparation of cell smears; DIG labeling of oligonucleotides with DIG-ddUTP; In situ hybridization using digoxigenin-labeled oligonucleotides. - [Read Identification of Single Bacterial Cells Using DIG-Labeled Oligonucleotides Protocol]

Category: PCR Protocols: In-situ PCR Protocols

In Situ Polymerase Chain Reaction: Past, Present, Future. Roche. - [Read In Situ Polymerase Chain Reaction Roche]

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: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for in situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates. - [Read In Situ Hybridization to Human Metaphase Chromosomes Using DIG-, Biotin-, or Fluorochrome-Labeled]