loss Protocols

Category: Genetics and Genomics: Cancer Genetics Protocols: Loss of Heterozygosity Protocols

DNA for analysis is purified using salt precipitation. The method is gentle, limits the breakage of the long chromosomal strands, and avoids the use of phenol and chloroform. It is suitable for use with cultured cells, breast tumor tissue that has been subjected to hormone receptor analysis, and blood samples. The loss of heterozygosity assay is performed using a multiplex PCR, in which one of each primer pair is labeled with a different fluorophor. - [Read A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome]

Category: Genetics and Genomics: Cancer Genetics Protocols

Investigators can utilize X chromosome inactivation (methylation) to determine the clonality status of a tumor or premalignant lesion in females. The technique is based on a methylation-sensitive restriction enzyme and analysis of a polymorphic locus on the X chromosome. Clonal cell populations will show "loss" of the non-methylated allele after restriction digest. The assay can be performed on DNA recovered from microdissected samples. Both frozen tissue and fixed-embedded tissue can be used. - [Read Clonality - X Chromosome Inactivation Assay Protocol]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to use the mtr gene as a selectable marker for loss or gain of function. - [Read How to Use the mtr Gene as a Selectable Marker for Loss or Gain of Function]

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

This method enables the culturing of thyroid cells without loss of differentiation and medium change. It is potentially useful for the long-term study of drug effects on the thyroid gland. - [Read Human Thyroid Culture Protocol]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

Protocol enables the culturing of thyroid cells without loss of differentiation and medium change.  It is potentially useful for the long-term study of drug effects on the thyroid gland. - [Read Human Thyroid Culture Protocol]

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

There are several strategies to visualize the antibody. For transmitted light microscopy, color development substrates for enzymes are often used. The antibody can be directly labeled with the enzyme. However, such a covalent link between an antibody and an enzyme might result in a loss of both enzyme and antibody activity. For these reasons several multistep staining procedures have been developed, where intermediate link antibodies are used. In this protocol use the Vectastain ABC-kit. - [Read Immunocytochemistry in Free-Floating Sections Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, etc. Protocol provides a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1–1.5 h. - [Read Laser-capture Microdissection Protocol]

Category: Genetics and Genomics: Cancer Genetics Protocols: Loss of Heterozygosity Protocols

This method is used to detect genomic DNA deletions in tumor cells. - [Read Loss of Heterozygosity Protocol]

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for preparing siliconized pipettes. Such pipettes minimize the loss of embryos or embryonic tissues during transfer. - [Read Preparing Siliconized Pipettes Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocols utilize T hybridomas to detect expression of peptide-MHC complexes, since these cells provide the most convenient, consistent, and flexible T cell readout systems for these purposes. If desired, antigen-specific T cell clones can be used in lieu of T hybridoma cells, but T cell clones often give poorer responses than T hybridomas to fixed APCs due to fixation-induced loss of costimulator function. - [Read Presenting Exogenous Antigen to T Cells Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

A powerful way to identify a mutation in the gene of interest and to test mutant plants for phenotypes that are predicted to result from loss of function of that gene is by PCR screening. Pools of insertion lines are screened using one primer corresponding to the gene of interest and one primer corresponding to the end of the insertion element. The synthesis of a product indicates the presence of an insertion in the gene of interest. - [Read Screening DNA Pools for T-DNA Insertions in Arabidopsis Genes Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

In an attempt to accurately measure DNA content with simultaneous preservation of cell surface markers, we have utilized gentle ethanol treatment techniques, which permeablize cells with minimal loss of surface antigen expression and antibody-antigen association. For some cell types, the presence of apoptotic cells based on reduced DNA content can also be detected. One such technique employs the addition of ethanol to cells previously resuspended in high concentrations of fetal bovine serum... - [Read Simultaneous Analysis of DNA Content and Surface Immunophenotype Protocol]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

No cell culture problem is as universal as that of culture loss due to contamination. All cell culture laboratories and cell culture workers have experienced it. Culture contaminants may be biological or chemical, seen or unseen, destructive or seemingly benign, but in all cases they adversely affect both the use of your cell cultures and the quality of your research. Contamination problems can be divided into three classes: Minor annoyances, Serious problems, Major catastrophes. - [Read Understanding and Managing Cell Culture Contamination Protocol]