3,522 results match your criteria Cold Spring Harbor protocols[Journal]


An RNA-Seq Protocol for Differential Expression Analysis.

Cold Spring Harb Protoc 2019 Apr 5. Epub 2019 Apr 5.

The Francis Crick Institute, NW1 1ST London, United Kingdom

Here we consider RNA-Seq, used to measure global gene expression through RNA fragmentation, capture, sequencing, and subsequent computational analysis. , with its large number of RNA-rich, synchronously developing, and accessible embryos, is an excellent model organism for exploiting the power of high-throughput sequencing to understand gene expression during development. Here we present a standard RNA-Seq protocol for performing two-state differential gene expression analysis (between groups of replicates of control and treated embryos) using Illumina sequencing. Read More

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http://dx.doi.org/10.1101/pdb.prot098368DOI Listing
April 2019
1 Read

RNA Interference and Small RNA Analysis.

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.top097436. Epub 2019 Apr 1.

Small silencing RNAs have provided powerful reverse genetics tools and have opened new areas of research. This introduction describes the use of RNAi to suppress expression of individual genes for loss-of-function analysis. It also summarizes methods for measuring specific and global changes in small RNA expression, as well as methods to inhibit the function of individual endogenous small RNA species such as miRNAs. Read More

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http://dx.doi.org/10.1101/pdb.top097436DOI Listing

Counterstaining, Mounting, and Photographing Stained Cells.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot099770. Epub 2019 Apr 1.

Counterstains and mounting media for immunohistology must be compatible with the detection method used. The results of cell-staining experiments can be stored on film or digitally. Read More

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http://dx.doi.org/10.1101/pdb.prot099770DOI Listing
April 2019
1 Read

Detecting Alkaline Phosphatase-Labeled Cells.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot099721. Epub 2019 Apr 1.

Different substrates are available for detection of cells labeled with alkaline phosphatase. Naphthol-AS-BI-phosphate (NABP)/New Fuchsin produces an intense red product that is insoluble in alcohols as well as aqueous solutions and is compatible with a range of histochemical stains. Bromochloroindoyl phosphate/nitro blue tetrazolium (BCIP/NBT) is the most commonly used of the chromogenic substrates for alkaline phosphatase. Read More

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http://dx.doi.org/10.1101/pdb.prot099721DOI Listing
April 2019
2 Reads

Detecting Horseradish Peroxidase-Labeled Cells.

Authors:
Scott J Rodig

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot099713. Epub 2019 Apr 1.

A range of substrates is available for detection of cells labeled with horseradish peroxidase (HRP). Diaminobenzidine (DAB) is the most commonly used substrate and one of the most sensitive. It yields an intense brown product that is insoluble in both water and alcohol. Read More

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http://dx.doi.org/10.1101/pdb.prot099713DOI Listing
April 2019
1 Read

RNAi in S2 Cells by siRNA Duplex or dsRNA Transfection.

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot097485. Epub 2019 Apr 1.

This protocol describes an efficient method to trigger RNAi in S2 cells by using transfection reagents to deliver siRNAs or dsRNAs. Read More

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http://dx.doi.org/10.1101/pdb.prot097485DOI Listing
April 2019
1 Read

RNAi in Mammalian Cells by siRNA Duplex Transfection.

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot097451. Epub 2019 Apr 1.

Long dsRNA cannot be used in most cultured mammalian cells because it triggers the interferon response, causing widespread changes in gene expression and apoptosis. This protocol describes a method for delivering into mammalian cells siRNA duplexes that are too short to elicit the sequence-nonspecific responses associated with long dsRNA. Read More

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http://dx.doi.org/10.1101/pdb.prot097451DOI Listing

Preparation of siRNA Duplexes.

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot097444. Epub 2019 Apr 1.

This protocol describes how to anneal synthetic sense and antisense siRNAs to form siRNA duplexes, as well as the analysis of siRNA duplexes using nondenaturing polyacrylamide gel electrophoresis. Read More

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http://dx.doi.org/10.1101/pdb.prot097444DOI Listing
April 2019
1 Read

Preparation of Polymerase Chain Reaction Template DNA from Mouse Tail Tissue.

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot092700. Epub 2019 Apr 1.

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). In this protocol, a small piece from the tip of the tail is removed and processed using hot sodium hydroxide and Tris (HotSHOT). Read More

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http://dx.doi.org/10.1101/pdb.prot092700DOI Listing

Isolation of High-Molecular-Weight DNA from Mouse Tail Tips.

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot092692. Epub 2019 Apr 1.

DNA samples are prepared from mouse tail tips by Proteinase K digestion and subsequently extracted. The resulting preparation is suitable for use in Southern blotting or polymerase chain reaction (PCR). Read More

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http://dx.doi.org/10.1101/pdb.prot092692DOI Listing
April 2019
1 Read

Generating a Three-Dimensional Genome from with Hi-C.

Cold Spring Harb Protoc 2019 Mar 18. Epub 2019 Mar 18.

Department of Medicine, University of California, San Diego, San Diego, California 92093

Hi-C is a sequencing-based method that captures three-dimensional (3-D) genome interactions by counting the interaction frequencies of pairs of genomic loci. This protocol describes the application of in situ Hi-C to the embryo. Briefly, after fixing embryos with formaldehyde, nuclei are isolated and chromatin is digested with a restriction enzyme. Read More

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http://dx.doi.org/10.1101/pdb.prot098343DOI Listing

Lipofection.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.top096248. Epub 2019 Mar 1.

In the past, cloned DNA was introduced into cultured eukaryotic cells chiefly by biochemical methods using either calcium phosphate or diethylaminoethyl (DEAE)-dextran. Lipid reagents are now preferred because of the high efficiency of transfection that can be obtained and because of the ability of this class of reagents to mediate transfection of all types of nucleic acids into a wide range of cell types. Read More

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http://dx.doi.org/10.1101/pdb.top096248DOI Listing
March 2019
2 Reads

Measurement of Radioactivity in Nucleic Acids.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot100933. Epub 2019 Mar 1.

Radioactive isotopes are used as tracers to monitor the progress of many reactions used to synthesize DNA and RNA. To calculate the efficiency of such reactions, it is necessary to measure accurately the proportion of the radioactive precursor incorporated into the desired product. An effective method to achieve this goal is differential precipitation of the nucleic acid products with trichloroacetic acid (TCA). Read More

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http://dx.doi.org/10.1101/pdb.prot100933DOI Listing

Spun-Column Chromatography.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot100594. Epub 2019 Mar 1.

This protocol describes the setup and use of Sephadex G-50 or Bio-Gel columns for the purification of DNA. Read More

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http://dx.doi.org/10.1101/pdb.prot100594DOI Listing
March 2019
1 Read

Random Priming: Labeling of Purified DNA Fragments by Extension of Random Oligonucleotides.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot100586. Epub 2019 Mar 1.

In this protocol, oligonucleotides that are heterogeneous in sequence serve as primers for the initiation of DNA synthesis on single-stranded templates. Labeled dNTPs ([α-P]dNTPs or biotin-, DIG-, or fluorescein-labeled dUTPs) are incorporated into the new DNA by the Klenow fragment of DNA polymerase I (Pol I). The method can be adapted to radiolabel DNA in slices cut from gels cast with low-melting-temperature agarose. Read More

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http://dx.doi.org/10.1101/pdb.prot100586DOI Listing

Transfected Dendritic Cell Immunizations.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot100073. Epub 2019 Mar 1.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). An efficient way of generating antibodies is to introduce antigens of interest into DCs and then inject them into the host. This will result in initiation of an antigen-specific immune response mediated by T-cell immunity. Read More

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http://dx.doi.org/10.1101/pdb.prot100073DOI Listing

Labeling Antibodies Using a Maleimido Dye.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot099291. Epub 2019 Mar 1.

Fluorophore-maleimide derivatives are effective for labeling sulfhydryl-containing molecules. Maleimide groups react with free thiols at pH 6.5-7. Read More

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http://dx.doi.org/10.1101/pdb.prot099291DOI Listing

Labeling Antibodies Using -Hydroxysuccinimide (NHS)-Fluorescein.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot099283. Epub 2019 Mar 1.

-Hydroxysuccinimide (NHS)-ester derivatives are among the most commonly used reagents for labeling proteins. The method described here can be adapted to use practically any NHS fluorophore. Generally, a fluorophore is covalently bound to a macromolecule such as an antibody and acts as a reporter molecule used to measure the presence of the macromolecule. Read More

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http://dx.doi.org/10.1101/pdb.prot099283DOI Listing

RNAi in S2 Cells by dsRNA Soaking.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot097477. Epub 2019 Mar 1.

This is a simple method for inducing RNAi in S2 cells by soaking the cells in medium containing dsRNAs. In comparison with transfection, soaking requires fewer procedures and avoids the cost of transfection reagents, making it the method of choice for high-throughput RNAi screening. Moreover, soaking avoids potential toxicity associated with transfection reagents. Read More

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http://dx.doi.org/10.1101/pdb.prot097477DOI Listing
March 2019
2 Reads

Preparation of dsRNAs for RNAi by In Vitro Transcription.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot097469. Epub 2019 Mar 1.

This is a simple and widely used method for the preparation of dsRNA using a polymerase chain reaction (PCR) template-based, in vitro transcription reaction. The resulting dsRNA is then used to trigger RNAi in an appropriate cell or organism. Read More

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http://dx.doi.org/10.1101/pdb.prot097469DOI Listing

Histochemical Staining of Cell Monolayers for β-Galactosidase.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot095422. Epub 2019 Mar 1.

There are several methods for assaying the success of transient transfections. If a plasmid expressing β-galactosidase was used, then this histochemical staining procedure is simple to perform and yields dependable results. The following method was designed for cells growing in 60-mm culture dishes. Read More

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http://dx.doi.org/10.1101/pdb.prot095422DOI Listing

DNA Transfection Mediated by Cationic Lipid Reagents.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot095414. Epub 2019 Mar 1.

Liposomal transfection reagents vary in their ability to transfect cell lines efficiently. Some are generalists, whereas others are best used with specific cell types. The nonliposomal FuGENE 6 and the cationic liposomal Lipofectamine 2000 are examples of reagents that can successfully transfect most adherent and suspension cell types (including several primary and hard-to-transfect cell types) with negligible toxicity and a minimal number of manipulations. Read More

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http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09541
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http://dx.doi.org/10.1101/pdb.prot095414DOI Listing
March 2019
4 Reads

Long and Accurate Polymerase Chain Reaction (LA PCR).

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot095158. Epub 2019 Mar 1.

The standard polymerase chain reaction (PCR) is easily capable of amplifying segments of DNA smaller than ∼3 kb in length-sufficient for most purposes, but not enough to amplify an entire mammalian gene, nor even a cDNA of average dimensions. Instead of full-length products, standard PCR amplification of longer templates generates variously sized truncated molecules that appear as unattractive smears on a gel. Long and accurate PCR (LA PCR) addresses the issue in part by using a mixture of two different thermostable DNA polymerases to catalyze the amplification reaction. Read More

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http://dx.doi.org/10.1101/pdb.prot095158DOI Listing

Preparation of Polymerase Chain Reaction Templates from Embryonic and Adult Mouse Tissue Samples.

Cold Spring Harb Protoc 2019 Mar 1;2019(3):pdb.prot094391. Epub 2019 Mar 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, yolk sac, ear punch, toe, or 1- to 2-mm (maximum 5 mm) tail samples for PCR analysis. Read More

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http://dx.doi.org/10.1101/pdb.prot094391DOI Listing
March 2019
1 Read

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
2 Reads

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
1 Read

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
2 Reads

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
6 Reads

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
8 Reads

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://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot09514
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http://dx.doi.org/10.1101/pdb.prot095141DOI Listing
February 2019
12 Reads

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
8 Reads

Mapping Chromatin Features of Embryos.

Cold Spring Harb Protoc 2019 Apr 1;2019(4):pdb.prot100263. Epub 2019 Apr 1.

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
April 2019
2 Reads

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
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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
10 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
9 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