3,498 results match your criteria Cold Spring Harbor protocols[Journal]


How to Win the Battle with RNase.

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.top101857. Epub 2019 Feb 1.

Because ribose residues carry hydroxyl groups in both the 2' and 3' positions, RNA is chemically much more reactive than DNA and is easy prey to cleavage by contaminating RNases-enzymes with various specificities that share the property of hydrolyzing diester bonds linking phosphate and ribose residues. Because RNases are released from cells following lysis and are present on the skin, constant vigilance is required to prevent contamination of glassware and bench tops and the creation of aerosols carrying RNase. The problem is compounded because there is no simple method to inactivate RNases. Read More

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http://dx.doi.org/10.1101/pdb.top101857DOI Listing
February 2019

Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method.

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.prot100479. Epub 2019 Feb 1.

The standard method to recover fragments of DNA from polyacrylamide gels is the "crush and soak" technique. The eluted DNA is generally free of contaminants that inhibit enzymes or that are toxic to transfected or microinjected cells. The method requires time but little labor and results in recovery of <30%-90%, depending on the size of the DNA fragment. Read More

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http://dx.doi.org/10.1101/pdb.prot100479DOI Listing
February 2019

Tandem Immunoaffinity Purification Using Anti-FLAG and Anti-HA Antibodies.

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.prot098657. Epub 2019 Feb 1.

The immunoaffinity purification of target proteins followed by the identification and characterization of associated proteins by mass spectrometry is a widely used technique. An immunoaffinity purification bears resemblance to a standard immunoprecipitation; however, the end product for mass spectrometric analysis in the femtomole (10) to attomole (10) range is required to be of exceptional purity. This high degree of sensitivity in detection renders it of extreme importance to eliminate most if not all of the nonspecific background proteins and can be achieved by performing a tandem affinity purification (TAP). Read More

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http://dx.doi.org/10.1101/pdb.prot098657DOI Listing
February 2019
1 Read

Cross-Linking Antibodies to Beads with Disuccinimidyl Suberate (DSS).

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.prot098632. Epub 2019 Feb 1.

This protocol describes the cross-linking of antibodies to either Protein A or G agarose beads using disuccinimidyl suberate (DSS), a bifunctional cross-linker capable of directly reacting with two different amines to form stable amide bonds. Proteins, including antibodies, generally display several primary amines in the side chains of lysine (K) residues and the amino terminus of each polypeptide that represent available potential targets for -hydroxysuccinimide (NHS)-ester cross-linking reagents. The antibody-bead cross-linking process generates a reusable resource of antibody and beads, commonly referred to as an antibody-specific resin, and can be repeatedly used for the immunoprecipitation of specific proteins if treated and stored correctly. Read More

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http://dx.doi.org/10.1101/pdb.prot098632DOI Listing
February 2019

Cross-Linking Antibodies to Beads Using Dimethyl Pimelimidate (DMP).

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.prot098624. Epub 2019 Feb 1.

This protocol describes the cross-linking of antibodies to either Protein A or G agarose beads using dimethyl pimelimidate (DMP). DMP contains an imidoester at each end of a 7-carbon spacer arm and forms an amidine bond with amino groups at alkaline pH; however, cross-linking is more efficient when performed at pH >8. DMP will react with primary amines; thus, it is important that the cross-linking procedure is conducted using nonamine-containing buffers. Read More

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http://dx.doi.org/10.1101/pdb.prot098624DOI Listing
February 2019

Nested Polymerase Chain Reaction (PCR).

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.prot095182. Epub 2019 Feb 1.

Nested polymerase chain reaction (PCR) is used in situations in which it is necessary to increase the sensitivity and/or specificity of PCR, for example, when amplifying a particular member of a polymorphic gene family or when amplifying a cDNA copy of an mRNA present at very low abundance in a clinical specimen containing a heterogeneous population of cell types. Nested PCR usually involves two sequential amplification reactions, each of which uses a different pair of primers. The product of the first amplification reaction is used as the template for the second PCR, which is primed by oligonucleotides that are placed internal to the first primer pair. Read More

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http://dx.doi.org/10.1101/pdb.prot095182DOI Listing
February 2019

Inverse Polymerase Chain Reaction (PCR).

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.prot095166. Epub 2019 Feb 1.

The standard polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two inward-pointing primers. In contrast, inverse PCR (also known as inverted or inside-out PCR) is used to amplify DNA sequences that flank one end of a known DNA sequence and for which no primers are available. Inverse PCR DNA involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. Read More

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http://dx.doi.org/10.1101/pdb.prot095166DOI Listing
February 2019

Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates.

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.prot095141. Epub 2019 Feb 1.

The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates include those with long homopolymeric runs, inverted repeats, or GC-rich tracts-such as those containing >60% G + C residues-that are found in the regulatory regions of many mammalian genes. Localized regions of templates rich in GC residues tend to fold into complex secondary structures that might not melt during the annealing phase of the PCR cycle. Read More

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http://dx.doi.org/10.1101/pdb.prot095141DOI Listing
February 2019

Preparation of Mouse Tissue Lysates for Polymerase Chain Reaction.

Authors:
William Perry

Cold Spring Harb Protoc 2019 Feb 1;2019(2):pdb.prot094383. Epub 2019 Feb 1.

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). This protocol can be used to prepare embryonic tissues or yolk sac samples for PCR analysis, but it is also useful for adult tissues (such as tail) that do not lyse completely by boiling in SDS/NaOH. Nonionic detergents are substituted for SDS because they interfere less with the polymerase reaction. Read More

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http://dx.doi.org/10.1101/pdb.prot094383DOI Listing
February 2019

Skin Grafting in : A Technique for Assessing Development and Immunological Disparity.

Authors:
Yumi Izutsu

Cold Spring Harb Protoc 2019 Jan 3. Epub 2019 Jan 3.

Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan

Skin grafting in the amphibian has been used to detect not only allogeneic antigens that differ by minor H antigens or by one MHC haplotype, but also to detect ontogeny-specific antigens (including both emerging adult- and disappearing larval-specific) during metamorphosis. To understand the mechanisms underlying allogeneic tolerance or immune responses against larval- and/or adult-specific antigens, a complete MHC homozygous, inbred strain is the most appropriate experimental model. The inbred J strain established in Japan is used here. Read More

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http://dx.doi.org/10.1101/pdb.prot099788DOI Listing
January 2019
5 Reads

Mapping Chromatin Features of Embryos.

Cold Spring Harb Protoc 2019 Jan 3. Epub 2019 Jan 3.

Developmental Biology Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom

Chromatin immunoprecipitation (ChIP) combined with genomic analysis provides a global snapshot of protein-DNA interactions in the context of chromatin, yielding insights into which genome loci might be regulated by the DNA-associated protein under investigation. This protocol is an update of a previous version and describes how to perform ChIP on intact or dissected embryos. The ChIP-isolated DNA fragments are suitable for both deep sequencing (ChIP-Seq) and quantitative polymerase chain reaction (ChIP-qPCR). Read More

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http://dx.doi.org/10.1101/pdb.prot100263DOI Listing
January 2019
1 Read

Analysis of DNA by Agarose Gel Electrophoresis.

Cold Spring Harb Protoc 2019 Jan 2;2019(1):pdb.top100388. Epub 2019 Jan 2.

Electrophoresis through agarose or polyacrylamide gels is used to separate, analyze, identify, and purify DNA fragments. The technique is simple, rapid to perform, and capable of resolving fragments of DNA that cannot be separated adequately by other procedures, such as density gradient centrifugation. The location of bands of DNA within the gel can be determined directly by staining with low concentrations of fluorescent intercalating dyes, such as ethidium bromide or SYBR Gold; bands containing as little as 20 pg of double-stranded DNA can then be detected by direct examination of the gel in ultraviolet (UV) light. Read More

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http://dx.doi.org/10.1101/pdb.top100388DOI Listing
January 2019
2 Reads

Agarose Gel Electrophoresis.

Cold Spring Harb Protoc 2019 Jan 2;2019(1):pdb.prot100404. Epub 2019 Jan 2.

This protocol describes steps for the preparation and running of agarose gels and for staining and visualization of DNA in gels using three dyes: ethidium bromide, SYBR Gold, and SYBR Green 1. Read More

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http://dx.doi.org/10.1101/pdb.prot100404DOI Listing
January 2019
1 Read

Preparing and Using Adjuvants.

Cold Spring Harb Protoc 2019 Jan 2;2019(1):pdb.prot100214. Epub 2019 Jan 2.

Nonspecific stimulators of the immune response are known as adjuvants. The judicious use of adjuvants is essential to induce a strong antibody response to soluble antigens. The most commonly used adjuvant for research work is Freund's adjuvant. Read More

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http://dx.doi.org/10.1101/pdb.prot100214DOI Listing
January 2019
1 Read

Protein A and Protein G Purification of Antibodies.

Cold Spring Harb Protoc 2019 Jan 2;2019(1):pdb.prot099143. Epub 2019 Jan 2.

Protein A and Protein G are immunoglobulin-binding proteins expressed in and sp., respectively, that have been adapted for use in purifying large amounts of IgG. They are available covalently attached to affinity resins such as 4% cross-linked agarose, making them suitable for low-pressure antibody isolation. Read More

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http://dx.doi.org/10.1101/pdb.prot099143DOI Listing
January 2019
1 Read

Purification of Antibodies: Diethylaminoethyl (DEAE) Chromatography.

Cold Spring Harb Protoc 2019 Jan 2;2019(1):pdb.prot099135. Epub 2019 Jan 2.

Because IgG from most species (other than rodents) tends to have an isoelectric point around neutral, two approaches can be used when separating IgG using diethylaminoethyl (DEAE) resins. When serum containing antibodies is applied to DEAE at a slightly acidic pH, the IgG flows through the column while most other serum proteins bind to the DEAE. This method is best performed using a batch method. Read More

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http://dx.doi.org/10.1101/pdb.prot099135DOI Listing
January 2019
1 Read

Saturation Mutagenesis by Codon Cassette Insertion.

Cold Spring Harb Protoc 2019 Jan 2;2019(1):pdb.prot097790. Epub 2019 Jan 2.

Saturation mutagenesis by cassette insertion introduces a library of site-specific changes into a specific DNA sequence within a target gene and is especially useful for analyzing the effect of specific residues on the structure and function of a protein. In this protocol, a set of 11 universal oligodeoxyribonucleotide cassettes is used to generate mutations. The major advantage of this method is that a single set of mutagenic codon cassettes can be used to insert codons encoding all possible amino acids at any predetermined site within a gene. Read More

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http://dx.doi.org/10.1101/pdb.prot097790DOI Listing
January 2019
1 Read

Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis).

Cold Spring Harb Protoc 2019 Jan 2;2019(1):pdb.prot097782. Epub 2019 Jan 2.

In this method, two oligonucleotide primers are hybridized to the same strand of a denatured double-stranded recombinant plasmid. One primer (the mutagenic primer) introduces the desired mutation into the target sequences, whereas the second primer carries a mutation that destroys a unique restriction site (called "unique site elimination," or USE) in the plasmid. The product of the first part of the method is a heteroduplex plasmid consisting of a wild-type parental strand and a new full-length strand that carries the desired mutation but no longer contains the unique restriction site. Read More

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http://dx.doi.org/10.1101/pdb.prot097782DOI Listing
January 2019
1 Read

Isolation of High-Molecular-Weight DNA from Mouse Yolk Sacs and the Like.

Cold Spring Harb Protoc 2019 Jan 2;2019(1):pdb.prot092726. Epub 2019 Jan 2.

Often, genotyping of mouse embryos is required, and a small part, such as the yolk sac, can be used for this purpose. Here, DNA samples are prepared from extra-embryonic tissues by digestion with Proteinase K and subsequent extraction. The yolk sac of mid-gestation or later-stage embryos provides a sufficient amount of DNA for Southern analysis. Read More

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http://dx.doi.org/10.1101/pdb.prot092726DOI Listing
January 2019
2 Reads

Mapping of In Vivo RNA-Binding Sites by Ultraviolet (UV)-Cross-Linking Immunoprecipitation (CLIP).

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.top097931. Epub 2018 Dec 3.

RNA "CLIP" (cross-linking immunoprecipitation), the method by which RNA-protein complexes are covalently cross-linked and purified and the RNA sequenced, has attracted attention as a powerful means of developing genome-wide maps of direct, functional RNA-protein interaction sites. These maps have been used to identify points of regulation, and they hold promise for understanding the dynamics of RNA regulation in normal cell function and its dysregulation in disease. Read More

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http://dx.doi.org/10.1101/pdb.top097931DOI Listing
December 2018
1 Read

Optical Transfection.

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.top096222. Epub 2018 Dec 3.

Strategies for the delivery of genes into eukaryotic cells fall into three categories: transfection by biochemical methods, transfection by physical methods, and virus-mediated transduction. "Optical transfection"-a physical transfection method-exploits the ability of light to create small transient pores in the plasma membrane of mammalian cells. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.top096222
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http://dx.doi.org/10.1101/pdb.top096222DOI Listing
December 2018
6 Reads

Sampling and Preparation of Rabbit Serum.

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.prot100255. Epub 2018 Dec 3.

Periodic test bleeds collected from immunized animals should be checked for the desired antibodies. For rabbits, test bleeds normally are performed from the marginal ear vein. Five to 10 mL can be collected conveniently and will provide more than enough serum for most tests. Read More

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http://dx.doi.org/10.1101/pdb.prot100255DOI Listing
December 2018
5 Reads

Preparing Antigens Using a Baculovirus Expression System.

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.prot100065. Epub 2018 Dec 3.

Baculovirus-produced recombinant proteins circumvent many of the issues that limit bacterial protein production. Baculoviruses contain double-stranded, circular, supercoiled DNA in a rod-shaped capsid. The viral life cycle includes three major phases: (1) early (or virus synthesis) phase (0. Read More

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http://dx.doi.org/10.1101/pdb.prot100065DOI Listing
December 2018
1 Read

Isolation of the RNA Cross-Linking Immunoprecipitation (CLIP) Tags, 5'-Linker Ligation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification, and Sequencing.

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.prot097972. Epub 2018 Dec 3.

This protocol describes purification of RNA cross-linking immunoprecipitation (CLIP) tags by proteinase K digestion of the cross-linked protein, addition of a 5' linker to the RNA tags, and amplification of the product by transcription-polymerase chain reaction (RT-PCR). Use of this protocol adds another important purification step: sizing of the PCR products to enrich for those derived from RNA originally cross-linked to the desired RNABP. Finally, sequencing of the PCR products is described. Read More

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http://dx.doi.org/10.1101/pdb.prot097972DOI Listing
December 2018
2 Reads

3'-Linker Ligation and Size Selection by SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP).

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.prot097964. Epub 2018 Dec 3.

This protocol describes the purification by denaturing polyacrylamide gel electrophoresis of RNA linkers for cross-linking immunoprecipitation (CLIP). Purification is necessary because if the 3' linker loses the puromycin blocking group, concatemerization of the 3' linker will occur during the 3' linker ligation reaction. In addition, truncated linkers make bioinformatic processing of the sequencing results more difficult than it need be. Read More

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http://dx.doi.org/10.1101/pdb.prot097964DOI Listing
December 2018
1 Read

Immunoprecipitation and SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP).

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.prot097956. Epub 2018 Dec 3.

This first part of this protocol is designed to optimize purification of the RNABP by immunoprecipitation for cross-linking immunoprecipitation (CLIP) experiments. The key variables to assess are the quality and quantity of antibody needed to immunoprecipitate most but not quite all of the RNABP (the titration will decrease nonspecific binding), and the tolerance of the antibody:antigen interaction to stringent wash conditions. The results of these experiments can be checked first by western blot, and subsequently using the pilot CLIP protocol described in the second half of this protocol. Read More

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http://dx.doi.org/10.1101/pdb.prot097956DOI Listing
December 2018
1 Read

Ultraviolet (UV) Cross-Linking of Live Cells, Lysate Preparation, and RNase Titration for Cross-Linking Immunoprecipitation (CLIP).

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.prot097949. Epub 2018 Dec 3.

One of the great advantages of RNA CLIP (cross-linking immunoprecipitation) is that RNA-protein complexes can be "frozen" in situ in live cells by ultraviolet (UV) irradiation. This protocol describes UV cross-linking of mammalian tissue culture cells or whole tissues. For the latter, the tissue is typically triturated to allow UV penetration. Read More

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http://dx.doi.org/10.1101/pdb.prot097949DOI Listing
December 2018
1 Read

In Vitro Screen to Identify Silent but Activatable (S/A) Integration Sites for a Tetracycline-Inducible Transgene in Mice.

Cold Spring Harb Protoc 2018 Dec 3;2018(12):pdb.prot092684. Epub 2018 Dec 3.

To obtain ubiquitous or simply widespread transgene expression from a single stable integrant transgene is quite challenging because the random genomic integration sites of transgenes may create expression variation or frequent silencing. The tetracycline (Tet)-inducible system requires two reliable working transgenes, one for the tetracycline transactivators (tTA or rtTA) and one for the responder transgene driven by the promoter. Therefore, the challenge of getting this system working properly is a serious prospect. Read More

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http://dx.doi.org/10.1101/pdb.prot092684DOI Listing
December 2018
1 Read

Fluorescent Cell Staining Methods for Living Embryos.

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.prot106021. Epub 2018 Nov 1.

Biology Department, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599;

The tardigrade was chosen as a model system in part because animals and embryos are optically clear at all stages, facilitating the visualization of events in living material. Here we report new methods for introducing fluorescent dyes into developing embryos, including methods for fluorescently marking mitochondria, lysosomes, membranes, and nuclei. The development of these techniques suggests approaches for attempting to introduce other molecules into embryos. Read More

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http://dx.doi.org/10.1101/pdb.prot106021DOI Listing
November 2018
1 Read

Total RNA Extraction from Tardigrades.

Authors:
Thomas C Boothby

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.prot102376. Epub 2018 Nov 1.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Purification of high-quality total RNA from specimens is essential for many molecular techniques. In tardigrades (water bears), disruption of the cuticle is an important step in obtaining a good yield that is representative of all tissues of the animal. As with all single-stranded RNA methods, sterile technique, proper storage conditions, and handling are required for maintaining the quality and integrity of the material. Read More

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http://dx.doi.org/10.1101/pdb.prot102376DOI Listing
November 2018
1 Read

Microinjection of dsRNA in Tardigrades.

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.prot102368. Epub 2018 Nov 1.

Department of Biology, Seattle Pacific University, Seattle, Washington 98119

Classical genetic analysis in the tardigrade is a challenge because these animals are parthenogens. The publication of the genome has facilitated the study of targeted genes by RNA interference (RNAi), a robust mechanism to disrupt gene function. This protocol describes microinjection of double-stranded RNA (dsRNA) in tardigrades using techniques adapted from protocols originally developed in A DNA template (either genomic or cDNA) is used to prepare dsRNA, to which T7 polymerase binding sites are added at the 5' end of each strand. Read More

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http://dx.doi.org/10.1101/pdb.prot102368DOI Listing
November 2018
1 Read

Embryonic In Situ Hybridization for the Tardigrade .

Authors:
Frank W Smith

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.prot102350. Epub 2018 Nov 1.

Biology Department, University of North Florida, Jacksonville, Florida 32224

In situ hybridization is a method for visualizing embryonic gene expression that is amenable to nonmodel systems. Here, an in situ hybridization protocol is presented for the tardigrade This method allows gene expression to be visualized directly and with fluorescence microscopy. Read More

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http://dx.doi.org/10.1101/pdb.prot102350DOI Listing
November 2018
1 Read

Embryonic Immunostaining for the Tardigrade .

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.prot102343. Epub 2018 Nov 1.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.

Immunostaining is a method used to visualize the localization of proteins in fixed tissue. Many antibodies are available that recognize specific proteins in a wide diversity of organisms, which makes this method ideal for investigating gene expression patterns in nonmodel animal systems. This protocol describes immunostaining for studies of embryogenesis in the tardigrade . Read More

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http://dx.doi.org/10.1101/pdb.prot102343DOI Listing
November 2018
1 Read

Live Imaging of Tardigrade Embryonic Development by Differential Interference Contrast Microscopy.

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.prot102335. Epub 2018 Nov 1.

Biology Department, University of North Carolina, Chapel Hill, North Carolina 27599;

The tardigrade was chosen as a model system in part because embryos and animals are optically clear at all stages, facilitating the viewing and filming of internal processes. Multiplane video recordings under differential interference contrast (DIC) microscopy have allowed early embryonic cell lineages to be reconstructed through seven rounds of division and have revealed invariant patterns of asymmetric cell divisions, nuclear migrations, and cell migrations. Here, we present a protocol for filming embryonic development of by DIC microscopy. Read More

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http://dx.doi.org/10.1101/pdb.prot102335DOI Listing
November 2018
1 Read

Desiccation of .

Authors:
Thomas C Boothby

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.prot102327. Epub 2018 Nov 1.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Many species of tardigrades can survive severe water loss, but different species tolerate different desiccation conditions. is able to survive desiccation after an initial period of slow drying, as described here. This protocol will likely work for other tardigrade species as well. Read More

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http://dx.doi.org/10.1101/pdb.prot102327DOI Listing
November 2018
1 Read

Laboratory Culture of .

Authors:
Robert McNuff

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.prot102319. Epub 2018 Nov 1.

Sciento, Whitefield, Manchester, M45 6TB, England, United Kingdom

I have reared a culture of the tardigrade for 30 years, since 1987. Here, I present my culture protocol. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot10231
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http://dx.doi.org/10.1101/pdb.prot102319DOI Listing
November 2018
5 Reads

The Emergence of the Tardigrade as a Model System.

Authors:
Bob Goldstein

Cold Spring Harb Protoc 2018 Nov 1;2018(11):pdb.emo102301. Epub 2018 Nov 1.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

The success of scientists in revealing biological mechanisms has depended in large part on choosing tractable model systems. In 1997, molecular phylogenetics revealed that two of biology's most tractable models- and -are much more closely related to each other than had been thought previously. I began to explore whether any of the little-studied members of this branch of the tree of life might serve as a new model for comparative biology that could make use of the rich and ongoing sources of information flowing from and research. Read More

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http://dx.doi.org/10.1101/pdb.emo102301DOI Listing
November 2018
2 Reads

Analysis and Normalization of Real-Time Polymerase Chain Reaction (PCR) Experimental Data.

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.top095000. Epub 2018 Oct 1.

In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. The fluorescent reporter is excited by light from the real-time PCR machine, a fluorescence-detecting thermocycler. By recording the amount of fluorescence emission at each cycle, the PCR can be monitored during the exponential phase when the first significant increase in the amount of PCR product correlates with the initial amount of target template. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.top095000
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http://dx.doi.org/10.1101/pdb.top095000DOI Listing
October 2018
7 Reads

Making Weak Antigens Strong: Cross-Linking Peptides to KLH with Maleimide.

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot100016. Epub 2018 Oct 1.

Haptens, which are small antigens such as peptides and drug compounds, are very weakly or nonimmunogenic by themselves and require the assistance of carrier proteins: complex molecules capable of eliciting a strong immune response in the host on injection. The haptens serve as epitopes for binding to the antibodies on the B-cell surface, and the carriers provide the MHC class II-T-cell receptor binding sites. Keyhole limpet hemocyanin (KLH) is one of the most widely used of such carrier proteins. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot10001
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http://dx.doi.org/10.1101/pdb.prot100016DOI Listing
October 2018
16 Reads

Immunoblotting: Transfer of Proteins from Gels to Membranes.

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot098442. Epub 2018 Oct 1.

Electrophoretic transfer of proteins from gels to membranes can be achieved either by complete immersion of a gel-membrane sandwich in a buffer (wet transfer) or by placing the gel-membrane sandwich between absorbent paper soaked in transfer buffer (semidry transfer). For the wet transfer, the sandwich is placed in a buffer tank with platinum wire electrodes. For the semidry transfer, the gel-membrane sandwich is placed between carbon plate or stainless steel electrodes. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09844
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http://dx.doi.org/10.1101/pdb.prot098442DOI Listing
October 2018
2 Reads

Resolving Proteins for Immunoblotting by Gel Electrophoresis.

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot098434. Epub 2018 Oct 1.

This protocol describes Tris/glycine SDS-polyacrylamide gel electrophoresis, also known as SDS discontinuous gel electrophoresis or the Laemmli electrophoresis system. The gel-casting unit is assembled and tested to make sure that there are no leaks. Ammonium persulfate and tetramethylethylenediamine are added to the separating monomer solution, and the bottom (separating) gel is poured and allowed to polymerize. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09843
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http://dx.doi.org/10.1101/pdb.prot098434DOI Listing
October 2018
10 Reads

Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot095042. Epub 2018 Oct 1.

This protocol describes a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using a two-enzyme, two-tube approach, carried out using either SYBR Green I or TaqMan chemistries. The protocol uses a PCR volume of 20 µL (although most manufacturers recommend 50-µL reactions). However, if the PCR target is not very abundant (i. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09504
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http://dx.doi.org/10.1101/pdb.prot095042DOI Listing
October 2018
2 Reads

Quantification of DNA by Real-Time Polymerase Chain Reaction (PCR).

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot095034. Epub 2018 Oct 1.

There are few differences between the experimental steps necessary for amplifying template DNA in a real-time thermocycler and a standard polymerase chain reaction (PCR). In real-time PCR, it is necessary, however, to optimize the concentration of primers and probe and to perform a standard curve. It is also important to consider the data analysis method that will be used. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09503
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http://dx.doi.org/10.1101/pdb.prot095034DOI Listing
October 2018
4 Reads

Constructing a Standard Curve for Real-Time Polymerase Chain Reaction (PCR) Experiments.

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot095026. Epub 2018 Oct 1.

It is essential to prepare a standard curve for every real-time polymerase chain reaction (PCR) experiment. This protocol is used to construct a standard curve in which the template concentration is unknown. Such a standard curve is suitable for optimization experiments and for performing relative quantification by the standard curve method. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09502
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http://dx.doi.org/10.1101/pdb.prot095026DOI Listing
October 2018
2 Reads

Optimizing Primer and Probe Concentrations for Use in Real-Time Polymerase Chain Reaction (PCR) Assays.

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot095018. Epub 2018 Oct 1.

Once primers and probes have been designed and obtained, it is necessary to optimize their concentration for each real-time polymerase chain reaction (PCR) assay. A set of PCRs is assembled in which the concentrations of forward and reverse primers are varied independently. Following amplification of the template DNA, amplification plots are compared. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09501
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http://dx.doi.org/10.1101/pdb.prot095018DOI Listing
October 2018
2 Reads

Immunofluorescent Staining of Whole-Mount Mouse Embryos.

Authors:
Allison Stewart

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot094037. Epub 2018 Oct 1.

Mouse embryos and fetal organs have no pigmentation except for the retinal pigmented epithelium of the eye at subsequent stages of development. This makes it difficult to visualize and photodocument embryonic structures using conventional light microscopy. This protocol is used to localize specific proteins in whole embryos or fetal organs using immunofluorescence. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09403
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http://dx.doi.org/10.1101/pdb.prot094037DOI Listing
October 2018
10 Reads

DAPI Staining of Whole-Mount Mouse Embryos or Fetal Organs.

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.prot094029. Epub 2018 Oct 1.

Mouse embryos and fetal organs have no pigmentation except for the retinal pigmented epithelium of the eye at subsequent stages of development. This makes it difficult to visualize and photodocument embryonic structures using conventional light microscopy. A simple method is provided here that uses fluorescent nuclear stains. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09402
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http://dx.doi.org/10.1101/pdb.prot094029DOI Listing
October 2018
14 Reads

Erratum: Electrophoretic Mobility Shift Assays for RNA-Protein Complexes.

Authors:
Donald C Rio

Cold Spring Harb Protoc 2018 Oct 1;2018(10):pdb.err106104. Epub 2018 Oct 1.

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.err106104
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October 2018
2 Reads

3C-Based Chromatin Interaction Analyses.

Cold Spring Harb Protoc 2018 Sep 4;2018(9):pdb.top097832. Epub 2018 Sep 4.

This introduction presents a molecular approach that uses formaldehyde cross-linking to investigate genome structure and function-chromosome conformation capture (3C). This approach allows us to determine the spatial proximity of distant functional genomic sites (by looping). 3C-based techniques to interrogate chromosome folding and long-range interactions between genomic sequences in vivo are detailed. Read More

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September 2018
4 Reads

Selective Agents for Stable Transfection.

Cold Spring Harb Protoc 2018 Sep 4;2018(9):pdb.top096230. Epub 2018 Sep 4.

A stable cell line is generated when transfected DNA undergoes integration into a chromosome by nonhomologous recombination. Cells that stably express selectable (e.g. Read More

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September 2018
1 Read