treatment 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: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Additional slide pre-treatment for mapping smaller insert clones for FISH. Sanger Institute. - [Read Additional slide pre-treatment for mapping smaller insert clones]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Removal of the 5'-phosphate groups from the internal termini of bacteriophage {lambda} arms is used to prevent self-ligation and suppress the background of nonrecombinant bacteriophages during cloning. - [Read Alkaline Phosphatase Treatment of Bacteriophage lambda Vector DNA 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: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

Simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA-content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin-embedded tissues, and deconvolution of DNA-content-frequency... - [Read Analysis of Cellular DNA Content by Flow Cytometry Protocol]

Category: Neuroscience Protocols

After section of the corticospinal (CS) tract in adult rats, neutralizing Nogo-A with monoclonal antibodies leads to enhancement of axonal re-growth and compensatory sprouting, accompanied by increased motor recovery.  The neutralization of Nogo-A represents a promising approach for therapy after lesion if its enhancement of functional recovery can be transposed to primates. - [Read Anti-Nogo-A Treatment Protocol]

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: BACs Bacterial Artificial Chromosome Protocols

Protocol uses the BIOPRIME reaction kit from GibcoBRL to prepare biotin-labelled BAC DNA which is detected using FITC-Avidin (Vector Labs, DCS grade). Reagents from other manufacturers may work equally well but have not been tested. Includes: Labeling of BAC clones; Ethanol precipitation; Hybridization; Post-hybridisation treatment / detection. - [Read BAC-FISH Protocol]

Category: Epigenetics and Methylation Protocols

Bisulfite Treatment of DNA Anderson Lab. Adapted from Frommer et.al. - [Read Bisulfite Treatment of DNA Anderson Lab]

Category: Epigenetics and Methylation Protocols: Bisulfite Treatment Methylation Assay Protocol

Adapted from Frommer et.al. Good protocol for bisulfite treatment of DNA. Includes tips on Methylation PCR for CpG methylation analysis. University of Texas M. D. Anderson Cancer Center - [Read Bisulfite Treatment of DNA for Methylation Analysis]

Category: Protein Protocols: Protein Extraction From Cells

Cell Lysate Extracts. Great protocols for cell lysis preparation from a variety of cell types. There are numerous methods of cell stimulation and lysis. For a given protein, Upstate’s Laboratories determine the specific treatment upon initial testing of its products. It is important to select the correct cell line, stimulation procedure (if any), and lysis protocol. Upstate. - [Read Cell Lysate Extracts]

Category: Cell Organelle Protocols: Nucleus Protocols

The key step is the lysis which solubilizes centrosomes away from nuclei by very low ionic strength lysis after treatment of cells with nocodazole and cytochalasin B. The released centrosomes are then centrifuged onto a Ficoll cushion (to avoid pelleting) and the interface between the lysate and the Ficoll is collected and the centrosomes are concentrated on a sucrose gradient. Fractions are assayed by spindown and double IF with 5051 serum and anti-tubulin and the pooled fractions are frozen... - [Read CHO Centrosome Prep Protocol]

Category: Cytogenetics Protocols: Comparative Genomic Hybridization CGH Protocols

Excellent Detailed CGH Comparative Genomic Hybridization Protocol. Squire Lab, Univ. Toronto. Includes: Labeling, Gel Interpretation, Slide Pre-Treatment and Denaturation, Probe Denaturation, Hybridization and Post Hybridization Washes and Detection, Tips - [Read Comparative Genomic Hybridization PDF]

Category: Epigenetics and Methylation Protocols: Bisulfite Treatment Methylation Assay Protocol

A detailed protocol for bisulfite treatment of DNA. DNA is first digested with a restriction enzyme. DNA is denauted at 97C for 5min. Bisulfite protocol is included. Also DNA purification using the Promega Wizard DNA Cleanup kit in indicated. Fan Lab. UCL - [Read Fan Lab Bisulfite Treatment of Genomic DNA]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Protocol for fixation and permeabilization of C. elegans. Protocol works for all stages except dauers (which won't open) and hypoclorite-treated eggs (which disintegrate). Luckily, hypochlorite treatment and fixation are by themselves sufficient to open eggs. - [Read Fixation and Permeabilization of C. elegans Protocol]

Category: General Research Protocols and Methods: Sterile Technique

Formaldehyde sterilization of tissue culture hoods. A bit toxic however effective. Dr.Dawson, Department Biochemistry, Univ. Nottingham Medical School. - [Read Formaldehyde Treatment of Tissue Culture Hoods]

Category: Cytogenetics Protocols

G-Banding observations. Includes: Slide storage; Fixative composition and trypsin treatment in G-banding; Banding of chemically aged slides. - [Read G-Banding Observations]

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

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocol]

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

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocol]

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

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocols]

Category: Cell Biology and Cell Culture: Troubleshooting Cell Culture Problems

Guide For Identifying And Correcting Common Cell Growth Problems. Corning. Surface Treatment Process, Problems Related to Technique, Problems Related to Incubators, Problems Related to Culture Media, Problem Solving Suggestions. - [Read Guide For Identifying And Correcting Common Cell Growth Problems]

Category: E Coli Protocols: E coli Nucleic Acid Protocols

Protocol for the isolation of total RNA from E. coli for Microarrays. Includes: Harvesting total RNA; DNase I treatment of RNA samples. - [Read Isolation of Total RNA form E. coli for Microarrays Protocol]

Category: Microarray Protocols: Microarray Analysis Protocols

Protocol for isolation of total RNA from E. coli for microarrays. Includes: Harvesting total RNA; Dnase I treatment of RNA samples. - [Read Isolation of Total RNA From E. Coli for Microarrays Protocol]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

No special treatment is required to prepare a lysate for the active collection. The following procedure should be used for long-term storage of lambda clones in the archival collections. The phage are diluted in media containing 7% DMSO and frozen at -80 degrees C. - [Read Long Term Lambda Phage Storage Protocol]

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

This protocol introduces a method for maintaining mammalian cell culture suitable for treatment with siRNA. Transfection of cultured cells with siRNAs (see Transfection of siRNA Duplexes into Mammalian Cells) and downstream analysis of the knockdown cells. - [Read Mammalian Cell Culture and Preparation of Cells for RNAi in 24-Well Plates Protocol]