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PCR-Based Screening of DNA Libraries Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4129

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

PCR-Based Screening of DNA Libraries Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4129

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]

PCR-Mediated Gene Disruption: One-Step Method Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4169

Category: PCR Protocols

DNA prepared by PCR-mediated gene disruption can be used to transform yeast in gene replacement experiments. This protocol uses two primers, tailed with approximately 50 nucleotides homologous to the gene of interest, that target insertion of the PCR product to that locus. Each primer ends with a universal sequence that is designed to amplify various selectable markers from plasmid templates. - [Read PCR-Mediated Gene Disruption: One-Step Method Protocol]

PEI Nanoparticles for Targeted Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4451

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes the preparation of polyethylenimine (PEI)/DNA nanoparticles for targeted gene delivery. This delivery strategy improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing uptake by nontarget cells. We describe here methods for the conjugation of targeting peptides to PEIs, formation of DNA complexes using the conjugated PEIs or nonconjugated PEIs together with targeting peptides, and cell transfection. - [Read PEI Nanoparticles for Targeted Gene Delivery Protocol]

Phosphorylating the 5' Termini of Oligonucleotides Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3880

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for phosphorylating the 5' termini of oligonucleotides. - [Read Phosphorylating the 5' Termini of Oligonucleotides Protocol]

Photoactivation-Based Labeling and In Vivo Tracking of RNA Molecules in the Nucleus - http://www.cshprotocols.org/cgi/content/abstract/2006/29/pdb.prot4600

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a method for observing and measuring the movement of RNA molecules in the nucleus of living mammalian cells. Caged fluorescein-labeled DNA oligonucleotides are introduced into living mammalian cells, where they demonstrably hybridize to complementary RNA. After site-specific photoactivation at desired sites within the cell, the RNA movements away from those sites are followed and digitally recorded using a rapid acquisition microscopy system. - [Read Photoactivation-Based Labeling and In Vivo Tracking of RNA Molecules in the Nucleus]

Picking Bacteriophage lambda Plaques Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3962

Category: Viral Manipulation Protocols: Phage Protocols

General method to pick and store plaques. - [Read Picking Bacteriophage lambda Plaques Protocol]

Picking Zygotes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4183

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Zygotes can be identified by their unique morphology. They can be easily separated away from nonmated cells using a micromanipulator. This method provides an alternative to the selection of diploid cells on a medium that prevents the growth of haploid parent cells. - [Read Picking Zygotes Protocol]

Plasmid Transformation of Yeast Colonies Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4146

Category: Yeast Protocols: Yeast Transformation Protocols

Protocol for plasmid transformation of Yeast colonies. Protocol can be used if high efficiency is not needed. - [Read Plasmid Transformation of Yeast Colonies Protocol]

Plate Assay for Carboxypeptidase Y in Yeast Cells Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4161

Category: Yeast Protocols: Yeast Genetics Protocols

Protocol for plate assay for carboxypeptidase Y in Yeast cells. - [Read Plate Assay for Carboxypeptidase Y in Yeast Cells Protocol]

Plating Bacteriophage Lambda Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3961

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes a method for generating isolated plaques from a stock of bacteriophage lambda. Each plaque derives from infection of a single bacterium by a single bacteriophage particle. Because each plaque contains the progeny of a single virus particle, the bacteriophages derived from a single plaque are essentially genetically identical to one another. - [Read Plating Bacteriophage Lambda Protocol]

Plating Bacteriophage M13 Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3993

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Bacteriophage M13 forms turbid plaques on lawns of male strains of E. coli. - [Read Plating Bacteriophage M13 Protocol]

Plating Cells for Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4650

Category: Cell Culture Protocols

Protocol describes a method for plating cells for microinjection onto etched coverslips. The coverslips for microinjection must be marked so that microinjected cells can be identified at time points after injection. - [Read Plating Cells for Microinjection Protocol]

Precipitation of Bacteriophage {lambda} Particles from Large-scale Lysates Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3966

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Bacteriophage {lambda} particles are recovered from bacterial lysates by precipitation with polyethylene glycol at high ionic strength. - [Read Precipitation of Bacteriophage {lambda} Particles from Large-scale Lysates Protocol]

Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in E coli Protocol - http://www.plantmethods.com/content/1/1/6

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli. Describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in E coli Protocol]

Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli - http://www.plantmethods.com/content/1/1/6

Category: Plant Biology Protocols: Plant Genetics Protocols

Describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli]

Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli - http://www.plantmethods.com/content/1/1/6

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli. Includes: Key steps in the EL250 RED-HR locus rescue and engineering procedure; Primer design and plasmid constructs; AtSTM gap-repair construct; Targeting construct backbone; Preparation of electrocompetent EL250 cells; Transformation of BAC F24o1 and induction of recombinogenic function in EL250; AtSTM locus rescue from BAC F24o1 by gap-repair HR. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli]

Preparation (Pulling) of Needles for Gene Delivery by Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4651

Category: Microinjection Protocols: Microinjection Equipment

This protocol contains methods for pulling microinjection needles using two different models of pipette pullers. The advantage of pulling needles in the laboratory is that a variety of different needle types can be pulled, depending on the samples and cells being injected. An added advantage is cost; once a pipette puller has been purchased, boxes of glass capillaries are inexpensive compared to premade microinjection needles. - [Read Preparation (Pulling) of Needles for Gene Delivery by Microinjection Protocol]

Preparation and Fractionation of Drosophila Embryo Extracts for the In Vitro Characterization of RNA - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4324

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes a method to prepare and fractionate Drosophila embryo extracts. These extracts can be used for the in vitro characterization of RNAi. - [Read Preparation and Fractionation of Drosophila Embryo Extracts for the In Vitro Characterization of RNA]

Preparation and High-efficiency Transformation of Competent E. coli - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3942

Category: Bacteria Transformation Protocols

Protocol generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/µg of superhelical plasmid DNA). - [Read Preparation and High-efficiency Transformation of Competent E. coli]

Preparation and Loading of Protein Samples for Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4656

Category: Microinjection Protocols: Microinjection of Proteins Protocols

his protocol provides methods for the preparation of protein samples and for loading them into pulled microinjection pipettes. Stock solutions of proteins are thawed, diluted (if desired), centrifuged at high speed to remove aggregates, and kept on ice until loading. Loading into micropipettes can be done using either a "front-loading" or a "backfilling" procedure. - [Read Preparation and Loading of Protein Samples for Microinjection Protocol]

Preparation and Microinjection of x-Rhodamine-Labeled Vimentin Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4676

Category: Cytoskeleton Protocols: Intermediate Filaments Protocols

Intermediate filaments (IF) are major cytoskeletal systems of vertebrate and many nonvertebrate cells whose expression is cell-type specific and developmentally regulated. This protocol describes the x-rhodamine labeling of one type of IF, vimentin, and a method for microinjection of the labeled vimentin into cultured cells. IF dynamics can then be examined with fluorescence microscopy. - [Read Preparation and Microinjection of x-Rhodamine-Labeled Vimentin Protocol]

Preparation and Transformation of Competent E. coli Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3942

Category: E Coli Protocols: E coli Transformation Protocols

Procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/µg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically. - [Read Preparation and Transformation of Competent E. coli Protocol]

Preparation and Transformation of Competent E. coli Using Calcium Chloride Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3932

Category: Bacteria Transformation Protocols

Protocol used to prepare batches of competent bacteria that yield 5 x 106 to 2 x 107 transformed colonies/µg of supercoiled plasmid DNA. - [Read Preparation and Transformation of Competent E. coli Using Calcium Chloride Protocol]

Preparation and Transformation of Competent E. coli Using Calcium Chloride Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3932

Category: E Coli Protocols: E coli Transformation Protocols

Protocol is is used to prepare batches of competent bacteria that yield 5 x 106 to 2 x 107 transformed colonies/µg of supercoiled plasmid DNA. - [Read Preparation and Transformation of Competent E. coli Using Calcium Chloride Protocol]

Articles (1-1 of 1)

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

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