libraries Protocols

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

Alkaline agarose gels are used chiefly to measure the size of first and second strands of cDNA (Construction of cDNA Libraries Stage 1: Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase) and to analyze the size of the DNA strand after digestion of DNA-RNA hybrids with nucleases such as S1. - [Read Alkaline Agarose Gel Electrophoresis Protocol]

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. If amplification should become necessary, it is best to expand the library by growth in liquid medium. - [Read Amplification and Storage of a Cosmid Library: Amplification in Liquid Culture Protocol]

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: DNA Protocols: cDNA Library Protocols

This stage achieves four goals: polishing the ends of double-stranded DNA, ligation of synthetic linkers or adaptors, digestion of the attached linkers to create cohesive termini, and preparing the cDNA for cloning. - [Read Attachment of Linkers or Adaptors for Construction of cDNA Libraries]

Category: RNA Protocols: cDNA Libraries Library

Includes procedures for cDNA screens using secondary and tertiary screening. Dr. Bart Frank Lab. - [Read Benton Davis Blots for cDNA Libraries]

Category: DNA Protocols: cDNA Library Protocols

Protocol for construction of cDNA library, stage 5: Fractionation of cDNA by Gel Filtration through Sepharose CL-4B. - [Read Construction of cDNA Libraries Stage 5: Fractionation of cDNA by Gel Filtration through Sepharose]

Category: DNA Protocols: Genomic DNA Library Protocols

This stage summarizes how to construct a cDNA library in a mammalian vector, using commercial kits. These kits are available from several manufacturers and generally include all the required reagents. - [Read Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening]

Category: DNA Protocols: cDNA Library Protocols

Here, the DNA-RNA hybrids synthesized in Stage 1 are converted into full-length double-stranded cDNAs. The primers for synthesis of second-strand cDNA are created by RNase H, which introduces nicks into the RNA moiety of the cDNA-mRNA hybrids. E. coli DNA polymerase I extends the newly created 3'-hydroxyl termini, using the first-strand cDNA as a template. - [Read Construction of cDNA Libraries Protocol]

Category: DNA Protocols: cDNA Library Protocols

The goal of this stage is to introduce methyl groups that will modify and protect naturally occurring EcoRI sites in the double-stranded cDNA. - [Read Construction of cDNA Libraries Protocol.]

Category: DNA Protocols: Cosmid Library Protocols

Protocol describes how to construct a library of 35-45-kb fragments of genomic DNA in the double cos site cosmid vector, SuperCos-1. The steps include: Linearization and dephosphorylation of SuperCos-1 DNA; Partial digestion of high-molecular-weight DNA with MboI; Dephosphorylation of high-molecular-weight genomic DNA; Ligation of cosmid arms to genomic DNA: Packaging and plating recombinants; Isolation and analysis of recombinant cosmids: Validation of the library. - [Read Construction of Genomic DNA Libraries in Cosmid Vectors Protocol]

Category: Yeast Protocols

Handling and using insertion libraries. Includes: Curing strains of endogenous 2-micron; mTn-lacZ/LEU2 insertion library; mTn-3xHA/lacZ insertion library; mTn-3xHA/GFP insertion library. - [Read Handling and Using Insertion Libraries]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

To identify the YAC subclones containing both a human insert and a portion of either the left or right arm of the pYAC4 vector. Identification of these clones is necessary in order to do YAC chromosome walking, and is also useful in the determination of whether a particular YAC clone has a contiguous human insert or whether a co-cloning event has occurred. Vector arm sequences are identified using pBR322 fragments from a BamHI-PvuII double digest. - [Read Identification of End Clones in YAC Subclone Libraries 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: RNA Protocols: cDNA Libraries Library

Adsroption of Anti-E. coli Antibodies, cDNA Library Screening, Frank Lab. Oklahoma Medical Research Foundation - [Read Immunoscreening of Lambda-GT11 Bacteriophage Libraries with Antibody Probes]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Procedure is used to prepare DNA simultaneously from many different types of samples or tissues.  Although the DNA is generally too small (approx.  80 kb) for efficient construction of genomic DNA libraries, it gives excellent results in Southern hybridizations and PCRs.  Cultured aneuploid mammalian cells (2 x 107, e.g., HeLa cells) yield 100 µg of DNA in a volume of 1 ml. - [Read Isolation of DNA from Mammalian Cells by Spooling Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Method of choice when large amounts of mammalian DNA are required, for example, for Southern blotting (Rapid Isolation of Mammalian DNA, Rapid Isolation of Yeast DNA, Southern Blotting: Capillary Transfer of DNA to Membranes) or for construction of genomic libraries in bacteriophage {lambda} vectors.  Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 x 107 cultured aneuploid mammalian cells (e.g., HeLa cells). - [Read Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. - [Read Isolation of Plant Transcription Factors Using a Modified Yeast One-Hybrid System]

Category: DNA Protocols: cDNA Library Protocols

The final stage of cDNA library construction involves optimizing ligation of the size-fractionated cDNA to bacteriophage {lambda} arms, followed by packaging and plating of the recombinant bacteriophages. - [Read Ligation of cDNA to Bacteriophage lambda Arms for the Construction of cDNA Libraries]

Category: RNA Protocols: cDNA Libraries Library

The Queensland Institute for Medical Research (QIMR) lab's method for automated sequencing for expressed sequence tags from Schistosoma japonicum and/or S. mansoni cDNA libraries constructed in the directional vectors lambda ZAP II and UNI ZAP XR. Paul Br - [Read Method for automated sequencing for expressed sequence tags from Schistosoma japonicum and/or S. man]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The latest generation of Promega cell-based assays includes luminescent and fluorescent chemistries to measure markers of cell viability, cytotoxicity and apoptosis, as well as to perform reporter analysis. Using these tools researchers can investigate how cells respond to growth factors, cytokines, hormones, mitogens, radiation, effectors, compound libraries and other signaling molecules. The protocols provided are guidelines for multiplexing cell-based assays & are intended as starting points. - [Read Multiplexing Cell Viability Assays Protocols]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Protocol is used to establish conditions for restriction enzyme digestion of eukaryotic genomic DNA that will generate fragments of a size appropriate for construction of genomic libraries.  To construct a genomic library, the average length of the starting genomic DNA should be at least eight times the capacity of the vector. - [Read Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

The results of the pilot experiment (Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions) are used to establish conditions for partial digestion of eukaryotic DNA in the large-scale reactions described here. - [Read Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Preparative Reactions Protocol]

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

PCR can be used to identify rare DNA sequences in DNA libraries by increasing the abundance of a particular sequence. This is accomplished by subdividing the original library into pools of decreased complexity and screening each pool or group of pools for a given DNA sequence. - [Read PCR-Based Screening of DNA Libraries Protocol]

Category: PCR Protocols

PCR can be used to identify rare DNA sequences in DNA libraries by increasing the abundance of a particular sequence.  This is accomplished by subdividing the original library into pools of decreased complexity and screening each pool or group of pools for a given DNA sequence.  A pool that contains the desired clone is subsequently subdivided into smaller pools, each of which is screened using the same PCR protocol that was used for the primary screen. - [Read PCR-Based Screening of DNA Libraries Protocol]

Category: Peptide Protocols: Phage Display Cloning Systems and Protocols

Ph.D. Peptide Display Cloning System - NEB. The Ph.D.â„¢ Peptide Display Cloning System facilitates the display of custom peptide libraries on the surface of bacteriophage M13 as coat protein fusions, creating a physical linkage between each displayed pep - [Read Ph.D. Peptide Display Cloning System - NEB]