filters Protocols

Category: DNA Protocols: Cosmid Library Protocols

Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. In this method of amplification, distortion of the library is rarely a problem because at no stage are bacteria containing different recombinant cosmids grown in competition with one another. - [Read Amplification and Storage of a Cosmid Library: Amplification on Filters Protocol]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Analysis of DNA Fragmentation Using the JAM Assay. By Shailaja Kasibhatla et al., The JAM assay is based on labeling nuclear DNA of cycling cells with [3H]thymidine and harvesting samples on glass fiber filters. Apoptosis will generate DNA fragments small enough to pass through the glass fiber filter, resulting in decreased radioactivity of the particular sample. Cell-mediated cytotoxicity or cell killing mediated by cytotoxic T lymphocytes (CTL) can also be measured by this technique. - [Read Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required)]

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

Bradford Protein Assay Spectrophotometry.  Includes spectrophotometry information and the Bradford protein assay: A spectrophotometer or colorimeter makes use of the transmission of light through a solution to determine the concentration of a solute within the solution.  A spectrophtometer differs from a colorimeter in the manner in which light is separated into its component wavelengths.  A spectrophotometer uses a prism to separate light and a colorimeter uses filters. - [Read Bradford Protein Assay Spectrophotometry]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

Protocol for cloning genes from a phage library. Includes: Titer and plate out phage; Lift plaques onto filters and prepare them for screening; Make a probe; Hybridize the probe to the filters; Wash the filters and expose to film; Purify putative plaques; Excise plasmid from the desired phage. - [Read Clone Genes From a Phage Library Protocol]

Category: Viral Manipulation Protocols: Phage Protocols

Using hybridization, it is possible to identify a single recombinant that carries the desired target sequence on a filter that carries the imprint of 15,000 or more plaques. - [Read Hybridization of Bacteriophage DNA on Filters Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how to identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with 32P-labeled double-stranded DNA rather than with antibodies. - [Read Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol]

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

Lipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. - [Read Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery Protocol]

Category: Bacterial Protocols

Alkali is used to liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to the filter by UV-cross-linking or baking under vacuum. - [Read Lysing Colonies and Binding of DNA to Filters Protocol]

Category: Radioactivity: Liquid Scintillation Counting

Excellent guide for Liquid Scintillation Counting. Includes protocols and methods for counting gel slices, SPECIAL SAMPLE PREPARATION PROTOCOLS: TLC Plates, protocol for Counting Samples on Cellulose-ester, Filters (MilliporeTM filters), Counting Tissue radioacitivity, Counting 14CO2, Samples in Polyacrylamide Gels. National Diagnostics. National Diagnostics Laboratory Staff. - [Read Principles and Applications of Liquid Scintillation Counting PDF]

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

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. - [Read Protocol: Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters]

Category: Nucleic Acid Purification Protocols

Protocol for purification of in vitro synthesized mRNA with Microcon or Centricon Centrifugal Filters. - [Read Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters Protocol]

Category: Antibody Protocols: Antibody Production Protocols

Antibodies directed against bacterial and phage-encoded proteins can be removed from sera by incubation with nitrocellulose filters impregnated with lysates of uninfected or phage-infected E. coli. - [Read Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening Protocol]

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

Run-on transcription monitors the regulation of transcription in isolated nuclei. Protocol includes: Preparation of Nuclei, Run-On Transcription Reaction, Alkaline Hydrolysis of Run-Off Transcripts, Preparation of Filters and Hybridization. Solutions included: DNase Solution, Hybridization Mix, Denhardt Solution (100X) etc. - [Read Run- On Transcription Protocol]

Category: DNA Protocols: Cosmid Library Protocols

Unamplified cosmid libraries are plated at high density onto 150-mm nitrocellulose or nylon filters and screened by hybridization - [Read Screening an Unamplified Cosmid Library by Hybridization: Plating the Library onto Filters Protocol]

Category: Bacterial Protocols

Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. - [Read Screening Bacterial Colonies by Hybridization: Intermediate Numbers Protocol]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

An expression library constructed in a bacteriophage {lambda} vector is plated on an appropriate E. coli strain in the absence of isopropylthio-ß-D-galactoside (IPTG). After 2-4 hours, the plates are moved to 37°C (to stabilize any fusion proteins that are temperature sensitive), and filters impregnated with IPTG are laid on top of the developing plaques. - [Read Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol]

Category: DNA Protocols: cDNA Library Protocols

A cDNA library constructed in a plasmid expression vector of the pUC, pUR, or pEX series is plated on agar medium and then replicated onto filters, which are transferred to plates containing IPTG. After 2-4 hours of induction, the colonies are lysed with chloroform and then screened with appropriate antibodies. - [Read Screening Expression Libraries Constructed in Plasmid Vectors Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

Protocol describes the preparation of subtracted cDNA probes by hybridization to an mRNA driver, followed by purification of the single-stranded radiolabeled cDNA by hydroxyapatite chromatography.  Before preparing the probe, it is a good idea to have filters (which contain the cDNA library to be screened) ready to hybridize. - [Read Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer Protocol]

Category: Viral Manipulation Protocols: Phage Protocols

Protocol is used to identify and isolate recombinants containing DNA sequences of interest. - [Read Transfer of Bacteriophage DNA from Plaques to Filters Protocol]