prior Protocols

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

Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels. - [Read Calibration of Becton Dickinson Flow Cytometers for Relative Fluorescence Intensity Measurements]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for Competitive RT-PCR.For quantifying mRNA, we use a competitive RT-PCR protocol with internal standard RNAs. These are added in a defined quantity to the RNA sample prior to the RT reaction. The resulting standard cDNA is coamplified with the s - [Read Competitive RT-PCR Protocol]

Category: Stem Cell Protocols

Protocol for the electroporation of ES cells. Cells are routinely passaged two days prior to electroporating. Usually one 10 cm plate at approximately 80% confluency will provide enough cells for 1-2 electroporations. - [Read Electroporation of ES Cells Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

This protocol describes a rapid PCR-based method for identifying targeted ES cell colonies prior to picking. It is based on DNA analysis of a small part of colonies pooled directly from selection plates. Only positive colonies are expanded. - [Read Genotyping Embryonic Stem (ES) Cell Colonies Prior to Picking Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis: RT-PCR and cDNA Synthesis FAQ

How can I eliminate DNA contamination from my RNA prior to RT-PCR? Invitrogen - [Read How can I eliminate DNA contamination from my RNA prior to RT-PCR?]

Category: Immunology: Immunoprecipitation Protocols

For many sources of antigens, one useful method of lysis is to treat cells with harsh, denaturing solutions to release most of the protein antigens, as described here. The lysates are then diluted to reduce the denaturing conditions to levels that are suitable for the formation of antibody-antigen complexes. The resulting solution is precleared prior to immunoprecipitation. - [Read Immunoprecipitation: Denaturing Lysis Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used. Includes information on: Permeabilization of Cells; In vitro Transcription Reaction (Run-off); Isolation of RNA; Preparation of Slot Blot Membrane for Hybridization; Hybridization of Nitrocellulose Membrane; TCA Precipitation to Determine Incorporation of [32P] GTP into Nucleic Acid - [Read In Vitro Transcription Assay (Run-off Assay) using Permeabilized Cells]

Category: Bacterial Protocols

Protocol was developed to isolate Wolbachia from adult Drosophila, but it can be adapted for other insects. In some insects leg removal prior to isolation facilitates hemolymph extrusion. - [Read Isolation of Live Bacteria from Adult Insects Protocol]

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

This protocol describes a method for quantitative measurement of DNA using propidium iodide (PI) staining and flow cytometry. PI stains all double-stranded regions of both DNA and RNA by intercalating between the stacked bases of the double helix. PI cannot penetrate an intact cell membrane; therefore, cells are fixed prior to staining. The ethanol-fixed cells can be stored unstained at 4°C for days, or even weeks, and then stained and analyzed. - [Read Measurement of DNA Content Using Propidium Iodide (PI) Staining of Fixed Whole Cells Protocol]

Category: Mass Spectrometry Protocols

Millipore's ZipTip Protocol. Intended for purifying and concentrating femtomoles to picomoles of protein, peptide or oligonucleotide samples prior to analysis, providing better data quality. Millipore. - [Read Millipore's ZipTip Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for Preparation of DNA-free RNA Prior to RT-PCR (with Deoxyribonuclease I) Fermentas - [Read Protocol for Preparation of DNA-free RNA Prior to RT-PCR Fermentas]

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Cell lines are reactivated and grown to a count of 1 x 108 cells. The cells are pelleted and stored frozen at -80 degrees C prior to DNA extraction. Donis-Keller. - [Read Reactivating Cell Lines and Cell Growth for DNA Preparation]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

This protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in RNase-free DNase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol describes a method for reverse transcriptase (RT) in situ PCR.  In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step.  The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in Rnase-free Dnase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for separating proteins using ion-exchange chromatography. Protocol details the practical considerations of an ion-exchange chromatography (IEC) experiment.  The choice of what type of ion exchanger to use, as well as the composition of the buffers used in this experiment, should be determined prior to beginning this protocol. - [Read Separating Proteins Using Ion-Exchange Chromatography Protocol]

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

This protocol describes a method for testing of new serum lots prior to use in cell culture. Serum lots vary considerably in their ability to support cell growth, and some lots even contain toxic or growth-inhibitory compounds. It is advantageous to test serum lots and purchase in large volume, both for cost benefit. - [Read Serum Testing for Mammalian Cell Culture Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Phage are streaked onto a medium to obtain an independent isolate prior to preparing a new lysate. This is done to reduce the likelihood of working with lysates which have become contaminated, and/or have accumulated mutations. - [Read Streaking Lambda Phages Protocol]