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Inverse PCR Protocol II - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3487

Category: PCR Protocols: Inverse PCR Protocols

Inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available. The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in the PCR. - [Read Inverse PCR Protocol II]

Isolating BAC DNA from Bacterial Cultures Using the NucleoBond System Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4390

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol describes isolation of BAC DNA from bacterial cultures using the commercial NucleoBond system. - [Read Isolating BAC DNA from Bacterial Cultures Using the NucleoBond System Protocol]

Isolating Germ Cells from the Genital Ridge - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4370

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol describes isolation of germ cells from the genital ridge of fetal mice from 11.5 days post coitum (dpc) onward. The germ cells can then be used for analysis, culture, or transplantation. - [Read Isolating Germ Cells from the Genital Ridge]

Isolating Germ Cells from the Genital Ridge Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4370

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

This protocol describes isolation of germ cells from the genital ridge of fetal mice from 11.5 days post coitum (dpc) onward. The germ cells can then be used for analysis, culture, or transplantation. - [Read Isolating Germ Cells from the Genital Ridge Protocol]

Isolating Individual Embryonic Stem (ES) Cell Colonies by Picking Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4410

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

This protocol describes a method for isolating individual ES cell colonies by picking after they have grown on selective medium. - [Read Isolating Individual Embryonic Stem (ES) Cell Colonies by Picking Protocol]

Isolating Mice Extraembryonic Membranes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4367

Category: Mouse Protocols: Mouse Embryology Protocols

Isolation of extraembryonic tissues allows one to study normal mouse development as well as the molecular basis of defects which cause fetal death. This protocol describes a method for isolating extraembryonic membranes from pregnant mice. - [Read Isolating Mice Extraembryonic Membranes Protocol]

Isolating Postimplantation Embryos: Early Neural-Fold-Stage Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4365

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations which cause postimplantation defects. This protocol describes a method for isolation of early neural-fold-stage embryos. - [Read Isolating Postimplantation Embryos: Early Neural-Fold-Stage Protocol]

Isolating Postimplantation Embryos: Early Primitive-Streak-Stage Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4363

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations which cause post-implantation defects. This protocol describes a method for isolating early primitive-streak-stage embryos. - [Read Isolating Postimplantation Embryos: Early Primitive-Streak-Stage Protocol]

Isolating Postimplantation Embryos: Early Somite-Stage Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4366

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations which cause postimplantation defects. This protocol describes a method for isolating early somite-stage embryos (~8.5 days post coitum [dpc]). - [Read Isolating Postimplantation Embryos: Early Somite-Stage Protocol]

Isolating Postimplantation Embryos: Late Primitive-Streak-Stage Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4364

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations which cause postimplantation defects. This protocol describes a method for isolating late primitive-streak-stage embryos (~7.5 days post coitum [dpc]). - [Read Isolating Postimplantation Embryos: Late Primitive-Streak-Stage Protocol]

Isolating Postimplantation Embryos: Prestreak-Stage Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4362

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Isolation of postimplantation-stage embryos allows one to study normal development as well as genetic mutations that cause post-implantation defects. This protocol describes a method for isolating prestreak-stage embryos (~5.5 days post coitum [dpc]). - [Read Isolating Postimplantation Embryos: Prestreak-Stage Protocol]

Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4009

Category: PCR Protocols: PCR Cloning Protocols

Protocol describes the use of vectorette PCR and single-site PCR to amplify the terminal sequences of genomic sequences cloned in high-capacity vectors such as PACs and YACs. - [Read Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors]

Isolation and Freezing of Primary Mouse Embryonic Fibroblasts (MEF) For Feeder Plates - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4482

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

This protocol describes the isolation of fibroblasts from mouse embryos. Mouse embryonic fibroblast (MEF) cells are used as a feeder layer for the culture of mouse embryonic stem (ES) cells to help maintain them as pluripotent stem cells. The inhibition of ES-cell differentiation provided by the MEF feeders appears to be due to their production of leukemia inhibitory factor (LIF). - [Read Isolation and Freezing of Primary Mouse Embryonic Fibroblasts (MEF) For Feeder Plates]

Isolation and Purification of Tetrahymena Nuclei Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4500

Category: Tetrahymena Protozoa Protocols

Protocol describes how to allow the isolation of nuclei from all stages of the Tetrahymena life cycle in high yield with a high degree of purity. This method gives highly purified populations of both micronuclei and macronuclei. - [Read Isolation and Purification of Tetrahymena Nuclei Protocol]

Isolation of BAC DNA from Large-scale Cultures Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4007

Category: BACs Bacterial Artificial Chromosome Protocols

The procedure for isolation of BAC DNA is scaled-up to accommodate 500-ml cultures, which, on average, yield 20-25 µg of purified BAC DNA. - [Read Isolation of BAC DNA from Large-scale Cultures Protocol]

Isolation of BAC DNA from Small-scale Cultures Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4006

Category: BACs Bacterial Artificial Chromosome Protocols

BAC DNAs are prepared from 5-ml cultures of BAC-transformed cells by a modification of the standard alkaline lysis method (Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation). The yield typically varies between 0.1 and 0.4 µg of BAC DNA. - [Read Isolation of BAC DNA from Small-scale Cultures Protocol]

Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot2936

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]

Isolation of DNA from Mammalian Cells by Spooling Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4037

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]

Isolation of DNA from Mammalian Cells Grown in 96-well Microtiter Plates Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3695

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

Protocol for isolation of DNA from mammalian cells grown in 96-well microtiter plates. Each well yields sufficient genomic DNA for several standard PCRs or for analysis in a single lane of a Southern hybridization. - [Read Isolation of DNA from Mammalian Cells Grown in 96-well Microtiter Plates Protocol]

Isolation of Fibroblasts from Chicken Embryos Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4475

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

This protocol describes the isolation of fibroblasts from chicken embryos. - [Read Isolation of Fibroblasts from Chicken Embryos Protocol]

Isolation of Fibroblasts from Mammalian Embryos Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4474

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

This protocol describes the isolation of fibroblasts from mouse, rat, and hamster embryos. - [Read Isolation of Fibroblasts from Mammalian Embryos Protocol]

Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4036

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]

Isolation of Plant Transcription Factors Using a Modified Yeast One-Hybrid System - http://www.plantmethods.com/content/2/1/3

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]

Isolation of Plasmid DNA from Yeast Cells: A Ten-Minute Preparation - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4150

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast DNA Isolation Protocols

Protocol describes the purification release of plasmid DNA from yeast cells. The resulting plasmid DNA can be used to transform Escherichia coli or yeast. - [Read Isolation of Plasmid DNA from Yeast Cells: A Ten-Minute Preparation]

Isolation of RNA from C. elegans Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4321

Category: C. elegans Protocols: C. elegans Genetics Protocols

Protocol describes an efficient approach to isolate RNA from Caenorhabditis elegans. - [Read Isolation of RNA from C. elegans Protocol]

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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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