works Protocols

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: DNA Protocols: Chromatin Immunoprecipitation Protocols

IP Wash buffer recipes and protocol for ChIP Assay. Protocol works with NIH 3T3, Friend, HeLa, Raji and CHO cells.. Farnham Lab - [Read Chromatin Immunoprecipitation (ChIPs) Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

Rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard Qiagen-Tip method that uses no organic extractions or columns. The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary. - [Read DNA Isolation From BAC & PAC Clones Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols

This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones.  It is a modification of a standard Qiagen-Tip method that uses no organic extractions or columns.  The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary. - [Read DNA Isolation From BAC & PAC Clones Protocol]

Category: Transfection Protocols: Stable Transfection Protocols

Pulsed electrical fields can be used to introduce DNA into a wide variety of animal cells. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate-DNA coprecipitation. But, as with other transfection methods, the optimal conditions for electroporating DNA into untested cell lines must be determined experimentally. - [Read DNA Transfection by Electroporation]

Category: DNA Protocols: EMSA DNA-Protein Protocols

DNaseI Footprinting Assay. Works well high affinity dna binding proteins. Breeden Lab. - [Read DNaseI Footprinting Assay]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Formamide can be used instead of proteinase K to dissociate bacteriophage {lambda} coat proteins from the viral {lambda}. The procedure is rapid and works best with large-scale preparations of bacteriophage {lambda}. - [Read Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Formamide Protocol]

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: DNA Protocols: EMSA DNA-Protein Protocols

This protocol works well for HSF DNA-binding activity experiments. - [Read Gel Mobility Shift Assay in PDF format]

Category: DNA Protocols: EMSA DNA-Protein Protocols

There are multiple variations to this protocol, but they find that this one works well in all cases tested. Mirmira Lab. - [Read General EMSA Protocol]

Category: Immunohistochemistry Protocols

Immunohistochemistry using the “milk method” with 0.01% triton X-100. This method works for the following antibodies: p27, cyclin D1, RbAP46. - [Read Immunohistochemistry Milk Method with 0.01% Triton X-100 Protocol]

Category: DNA Protocols: EMSA DNA-Protein Protocols

A non-radioactive electrophoretic mobility which works quite well using biotin and streptavidin detection. Non-specific and specific competititor, oligo labeling, Binding Reaction. Pierce - [Read Introduction to the EMSA (Gel Shift) Technique]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Polyacrylamide Gels Protocols

The "crush and soak" method, which works best for DNAs <1 kb in size, can be used to recover both singleand double-stranded DNAs from polyacrylamide gels.  The yield of eluted DNA varies from <30% to >90% depending on the size of the DNA fragment. - [Read Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method Protocol]

Category: DNA Protocols: DNA Extraction Protocols

Also works for onions. Duration ~40 minutes. DNA Isolation with very easily accessible household solutions. Great for school classes. science-projects.com - [Read Isolation of DNA from Strawberries and Raspberries]

Category: Molecular Imaging: Confocal Microscopy Protocols and Information

LRSM Confocal Microscope Home Page. Information on use and how confocal microscopy works. - [Read LRSM Confocal Microscope Home Page]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Protocol for PCR genotyping from tail DNA. This protocol works well for a variety of genes and primer pairs including Tg and KO alleles. Oligonucleotide melting temperatures between 60° and 65° seem to work well. - [Read PCR Genotyping from Tail DNA Protocol]

Category: Bacteria Transformation Protocols

Protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/µg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C. - [Read Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells Protocol]

Category: E Coli Protocols: E coli Transformation Protocols

Protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/µg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C. - [Read Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells Protocol]

Category: C. elegans Protocols

Protocol was designed to rapidly generate small scale cytosolic extracts of C. elegans for Western or IP (has not been tested for RNA work). The protocol works well for between 50 to 5000 worms and has not been extensively tested on larger a scale, though it should work. Includes: Collection; Sonication; Clearing lysate; Immunoprecipitation. - [Read Preparation of Worm Extracts by Sonication Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols

This method works well to assess cell cycle distribution of whole cell populations. This method can also be used to assess the cell cycle distribution of GFP transfected cells however, the EtOH step is generally not sufficient to keep GFP in the cell. - [Read Preparing Cells for PI/FACS (cell cycle) Analysis Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

A messy but reliable technique that works well for DNAs ranging in size from 200 bp to >50 kb. - [Read Recovery of DNA from Agarose and Polyacrylamide Gels: Electroelution into Dialysis Bags Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Polyacrylamide Gels Protocols

A messy but reliable technique that works well for DNAs ranging in size from 200 bp to >50 kb. - [Read Recovery of DNA from Agarose and Polyacrylamide Gels: Electroelution into Dialysis Bags Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

A fragment of gel containing a band of DNA is excised and digested with agarase, which hydrolyzes the polymer to disaccharide subunits.  The released DNA is then purified by phenol extraction and ethanol precipitation.  The method works well for DNAs ranging in size from <5 kb to >20 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Enzymatic Digestion with Agarase Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

DNA fragments separated by electrophoresis through gels cast with low-melting-temperature agarose are recovered by melting the agarose and extracting the resulting solution with phenol:chloroform.  The protocol works best for DNA fragments ranging in size from 0.5 kb to 5 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction Protocol]

Category: Genetics and Genomics: Cancer Genetics Protocols

SAGE is a new method that has been invented at Johns Hopkins University in USA to give scientists an overview of a cell’s complete gene activity. It works by capturing RNAs, identifying them and counting them. By comparing different types of cells, the researchers hope to generate profiles that will help them understand healthy cells and what goes wrong during diseases. Includes: How SAGE works and Steps of SAGE. - [Read Serial Analysis Of Gene Expression (SAGE)]