Showing papers in "Current protocols in molecular biology in 2001"
TL;DR: This procedure is effective in producing digestible chromosomal DNA from a variety of gram-negative bacteria, all of which normally produce large amounts of polysaccharides.
Abstract: Most protocols for the preparation of bacterial genomic DNA consist of lysis, followed by incubation with a nonspecific protease and a series of extractions prior to precipitation of the nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in removing exopolysaccharides which can interfere with the activity of enzymes such as restriction endonucleases and ligases. In this unit, however, the protease incubation is followed by a CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein, effectively removing both in the subsequent emulsification and extraction. This procedure is effective in producing digestible chromosomal DNA from a variety of gram-negative bacteria, all of which normally produce large amounts of polysaccharides. If large amounts of exceptionally clean DNA are required, the procedure can be scaled up and the DNA purified on a CsCl gradient, as described in the alternate protocol.
1,992 citations
TL;DR: This unit describes the isolation of MEFs and the use of g-irradiation or mitomycin C to produce inactivated feeders suitable for the culture of ES cells.
Abstract: Mitotically inactive mouse embryo fibroblasts (MEFs) are commonly used as feeder layers to prevent the differentiation of mouse embryonic stem (ES) cells. This unit describes the isolation of MEFs and the use of g-irradiation or mitomycin C to produce inactivated feeders suitable for the culture of ES cells.
130 citations
TL;DR: In this paper, the authors describe a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.
Abstract: This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield. The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. Once assembled, the mixture is cycled many times (usually 30) through temperatures that permit denaturation, annealing, and synthesis to exponentially amplify a product of specific size and sequence. The PCR products are then displayed on an appropriate gel and examined for yield and specificity. Recommended optimization conditions are included.
129 citations
TL;DR: EP-PCR takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations.
Abstract: Error-prone PCR (EP-PCR) is the method of choice for introducing random mutations into a defined segment of DNA that is too long to be chemically synthesized as a degenerate sequence. Using EP-PCR, the 5' and 3' boundaries of the mutated region may be defined by the choice of PCR primers. Accordingly, it is possible to mutagenize an entire gene or merely a segment of a gene. The average number of mutations per DNA fragment can be controlled as a function of the number of EP-PCR doublings performed. The EP-PCR technique described here is for a 400-bp sequence, and an Alternate Protocol is for a library. EP-PCR takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations.
101 citations
TL;DR: One‐hybrid screening in yeast is a powerful method to rapidly identify heterologous transcription factors that can interact with a specific regulatory DNA sequence of interest (the bait sequence) through in vivo reconstitution of a transcriptional activator that turns on expression of a reporter gene.
Abstract: One-hybrid screening in yeast is a powerful method to rapidly identify heterologous transcription factors that can interact with a specific regulatory DNA sequence of interest (the bait sequence). In this technique, the interaction between two proteins (bait and prey) is detected via in vivo reconstitution of a transcriptional activator that turns on expression of a reporter gene. Detection is based on the interaction of a transcription factor (prey) with a bait DNA sequence upstream of a reporter gene. To ensure that DNA binding results in reporter-gene activation, cDNA expression libraries are used to produce hybrids between the prey and a strong trans-activating domain. The advantage of cloning transcription factors or other DNA-binding proteins via one-hybrid screenings, compared to biochemical techniques, is that the procedure does not require specific optimization of in vitro conditions.
78 citations
TL;DR: The ligation-based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.
Abstract: This unit describes how PCR can be used to exponentially amplify segments of DNA located between two specified primer hybridization sites. A single-sided PCR method is used that initially requires specification of only one primer hybridization site; the second is defined by the ligation-based addition of a unique DNA linker. This linker, together with the flanking gene-specific primer, allows exponential amplification of any fragment of DNA. Because a defined, discrete-length sequence is added to every fragment, complex populations of DNA such as sequence ladders can be amplified intact with retention of single-base resolution. The ligation-based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.
72 citations
TL;DR: In this article, two basic protocols for introducing base changes into specific DNA sequences are described, the first describes the incorporation of a restriction site and the second details the generation of specific point mutations.
Abstract: This unit contains two basic protocols for introducing base changes into specific DNA sequences. The first describes the incorporation of a restriction site and the second details the generation of specific point mutations. An Alternate Protocol describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.
60 citations
TL;DR: This unit describes the simplest and most generally useful of the pulsed-field techniques, field inversion electrophoresis, which can be tuned to resolve molecules from 10 to 2000 kb (or more with specialized equipment).
Abstract: DNA molecules longer than 25 kb are poorly resolved by standard agarose gel electrophoresis. These longer molecules can be resolved using several techniques that periodically change the direction of the electric field in the gel. This unit describes the simplest and most generally useful of the pulsed-field techniques, field inversion electrophoresis, which can be tuned to resolve molecules from 10 to 2000 kb (or more with specialized equipment). To resolve molecules beyond the range of field inversion, it is necessary to use some sort of field-angle alternation electrophoresis such as CHEF (contour-clamped homogeneous electric field; described in an Alternate Protocol). A method is also provided for preparing high-molecular-weight DNA samples and size markers embedded in agarose blocks.
46 citations
TL;DR: This unit includes instructions for the various salt dialysis schemes (step versus gradient), which are accompanied by protocols for purification of core histones from bacteria and preparation of DNA for the nucleosomal arrays.
Abstract: Because it is convenient to assemble nucleosomal templates through salt dialysis, large amounts of chromatin complexes can be made easily and in a short amount of time. This unit includes instructions for the various salt dialysis schemes (step versus gradient), which are accompanied by protocols for purification of core histones from bacteria and preparation of DNA for the nucleosomal arrays. Electrophoretic procedures to analyze the reconstituted complexes are also included.
46 citations
TL;DR: Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed‐field gel electrophoresis (PFGE).
Abstract: Large-insert genomic libraries are necessary for physical mapping of large chromosomal regions, for isolation of complete genes, and for use as intermediates in DNA sequencing of entire genomes. Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, procedures for embedding total genomic DNA from lymphocytes or animal tissue cells are also provided. Other protocols detail partial digestion of genomic DNA with MboI or with a combination of EcoRI endonuclease and EcoRI methylase (including methods for optimizing the extent of digestion), and subsequent size fractionation by preparative PFGE. Finally, the isolation of BAC and PAC plasmid DNA for analyzing clones is also presented.
44 citations
TL;DR: This appendix lists recipes for common reagents and also describes equipment that is typically found in a molecular biology laboratory.
Abstract: This appendix lists recipes for common reagents and also describes equipment that is typically found in a molecular biology laboratory.
[...]
TL;DR: In the protocols in this unit, antigen is prepared for injection either by emulsifying an antigen solution with Freunds adjuvant or by homogenizing a polyacrylamide gel slice containing the protein antigen.
Abstract: In the protocols in this unit, antigen is prepared for injection either by emulsifying an antigen solution with Freunds adjuvant or by homogenizing a polyacrylamide gel slice containing the protein antigen. Mice are immunized at 2- to 3-week intervals. Test bleeds are collected 7 days after each booster immunization to monitor serum antibody levels. Mice are chosen for hybridoma fusions when a sufficient antibody titer is reached.
TL;DR: Methods for enzymatic amplification of RNA by the polymerase chain reaction (RT-PCR) are highlighted in this unit, especially useful for rare RNAs because all steps are performed under optimal conditions, thereby maximizing efficiency and recovery.
Abstract: Methods for enzymatic amplification of RNA by the polymerase chain reaction (RT-PCR) are highlighted in this unit The is especially useful for rare RNAs because all steps (annealing, reverse transcription, and amplification) are performed under optimal conditions, thereby maximizing efficiency and recovery These are also important considerations when amplifying heterogeneous RNA populations or large RNAs Although there are many commercially available kits for this procedure, the protocols listed here are good solid protocols that will work and that have withstood the test of time
TL;DR: This unit presents characteristics and reaction conditions of the DNA‐dependent DNA polymerases, including E. coli DNA polymerase I, Klenow fragment of E. Escherichia coli, T4DNA polymerase, native and modified T7 DNA polymeras, and Taq DNA polymerasing.
Abstract: This unit presents characteristics and reaction conditions of the DNA-dependent DNA polymerases, including E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, native and modified T7 DNA polymerase, and Taq DNA polymerase.
TL;DR: One of the most common methods for culturing ES cells using mouse embryo fibroblast feeder layers and recombinant leukemia inhibitory factor (LIF) to prevent differentiation is described.
Abstract: Mouse embryonic stem (ES) cells are used to generate mouse mutants by gene targeting and blastocyst-mediated transgenesis. ES cells must be cultured under conditions that prevent differentiation to maintain their ability to transmit altered alleles by contributing to the germ line. This unit describes one of the most common methods for culturing ES cells using mouse embryo fibroblast (MEF) feeder layers and recombinant leukemia inhibitory factor (LIF) to prevent differentiation.
TL;DR: This unit describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation.
Abstract: This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages A describes preparation of an E coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding
TL;DR: This unit provides protocols in which the viral particles are either pelleted or centrifuged onto a sucrose density step gradient, and how small volumes of virus stock can be concentrated by centrifugation through a filter that allows only small molecules to pass.
Abstract: For some applications, such as infection of cells in vivo, it is necessary to concentrate retrovirus stocks in order to increase their titer. Because viruses are macromolecular structures, they can be concentrated fairly easily by a relatively short centrifugation step. This unit provides protocols in which the viral particles are either pelleted or centrifuged onto a sucrose density step gradient. An alternate protocol details how virions can be precipitated using polyethylene glycol or ammonium sulfate, and the resulting precipitate collected by centrifugation. After resuspension of the precipitates, the virions can be used directly, or further purified either by sedimentation onto sucrose density gradients, or by column filtration as described here. Perhaps most simply of all, an alternate procedure describes how small volumes of virus stock can be concentrated by centrifugation through a filter that allows only small molecules to pass.
TL;DR: A modification to the Basic Protocol--inclusion of formamide in the sequencing gel--is designed to overcome gel compressions arising from secondary structure in the sequences products during gel electrophoresis.
Abstract: The accuracy of DNA sequence determination depends largely upon resolution of the sequencing products in denaturing polyacrylamide gels. This unit provides a detailed description of the setup, electrophoresis, and processing of such gels. In general, the gels required for DNA sequencing are 40-cm long, of uniform thickness, and contain 4% to 8% acrylamide and 7 M urea. Modifications of this protocol increase the length of readable sequence information which can be obtained from a single gel (i.e., forming the gel with wedge-shaped spacers to create a field gradient, or incorporating a buffer gradient, an electrolyte gradient, or an acrylamide step gradient into the gel). A modification to the Basic Protocol--inclusion of formamide in the sequencing gel--is designed to overcome gel compressions arising from secondary structure in the sequencing products during gel electrophoresis. A discussion of acrylamide concentrations and electrophoresis conditions is included in the Commentary.
TL;DR: This unit describes how differential display techniques allow the identification and subsequent isolation of differentially expressed genes that requires no knowledge of sequences, but rather PCR amplification using arbitrary oligonucleotides and high resolution polyacrylamide gel electrophoresis.
Abstract: Formerly unit Unavailable , this unit describes how differential display techniques allow the identification and subsequent isolation of differentially expressed genes that requires no knowledge of sequences, but rather PCR amplification using arbitrary oligonucleotides and high resolution polyacrylamide gel electrophoresis.
TL;DR: This unit describes the use of gels containing the nonionic detergent Triton X-100, referred to as Trit on/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones.
Abstract: Due to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X-100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.
TL;DR: This unit describes the strategies for generating and manipulating suitable ends on the PCR fragments and the efficiency of direct cloning of PCR products can be improved by generating suitable Ends on the amplified fragments.
Abstract: It is often desirable to clone PCR products to establish a permanent source of cloned DNA for hybridization studies, to obtain high-quality DNA sequencing results, or to separate products when PCR amplification yields a complex mixture. The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments. This unit describes the strategies for generating and manipulating suitable ends on the PCR fragments.
TL;DR: The method uses the polymerase chain reaction (PCR) to amplify cDNAs after each subtraction to prepare tracer and driver for the next subtraction, which uses double‐stranded cDNA (ds cDNA) as both tracers and driver.
Abstract: Subtractive cloning is a powerful technique that allows isolation and cloning of mRNAs differentially expressed in two cell populations. In the generalized subtraction scheme the cell types to be compared are the [+] or tracer cells and the [-] or driver cells, where mRNAs expressed in the tracer and not the driver are isolated. Briefly, tracer nucleic acid (cDNA or mRNA) from one cell population is allowed to hybridize with an excess of complementary driver nucleic acid from a second cell population to ensure that a high percentage of the tracer forms hybrids. Hybrids that form include sequences common to both cell populations. Hybrids between the tracer and driver, and all driver sequences, are removed in the subtraction step. The unhybridized fraction is enriched for sequences that are preferentially expressed in the tracer cell population. The method described in this unit uses double-stranded cDNA (ds cDNA) as both tracer and driver. Reciprocal subtractions are performed between two cell populations, A and B: that is, genes preferentially expressed in A more than in B are isolated, as are genes expressed preferentially in B more than in A. The method uses the polymerase chain reaction (PCR) to amplify cDNAs after each subtraction to prepare tracer and driver for the next subtraction. The progress of subtraction is monitored by slot blot hybridization. Differentially expressed cDNA sequences are used to construct a subtracted cDNA library.
TL;DR: This unit provides comprehensive protocols for the use of insertional libraries generated by shuttle mutagenesis, and a method by which Cre/lox recombination may be used to reduce the transposon in yeast to a small insertion element encoding an epitope tag is described.
Abstract: This unit provides comprehensive protocols for the use of insertional libraries generated by shuttle mutagenesis. From the basic protocol, a small aliquot of insertional library DNA may be used to mutagenize yeast, producing strains containing a single transposon insertion within a transcribed and translated region of the genome. This transposon-mutagenized bank of yeast strains may be screened for any desired mutant phenotype. Alternatively, since the transposon contains a reporter gene lacking its start codon and promoter, transposon-tagged strains may also be screened for specific patterns of gene expression. Strains of interest may be characterized by vectorette PCR (protocol provided) in order to locate the precise genomic site of transposon insertion within each mutant. A method by which Cre/lox recombination may be used to reduce the transposon in yeast to a small insertion element encoding an epitope tag is described. This tag serves as a tool by which transposon-mutagenized gene products may be analyzed further (e.g., localized to a discrete subcellular site).
TL;DR: A procedure prepares PCR products for use as templates for characterizing unlabeled product by genomic sequencing and chemical sequencing of end-labeled products are also presented.
Abstract: PCR products can be sequenced using either the dideoxy (Sanger) or chemical (Maxam-Gilbert) approaches. In the dideoxy methods presented here, the target sequence is amplified and an excess of one strand of the target sequence (relative to its complement) is then generated by "asymmetric PCR," where one primer is present in vast excess over the other. This single-stranded product serves as the template for conventional dideoxy sequencing methods. Another procedure prepares PCR products for use as templates fes for characterizing unlabeled product by genomic sequencing and chemical sequencing of end-labeled products are also presented.
TL;DR: Some antigen-antibody complexes can be dissociated in the presence of a combination of a low-molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt and seem to be generally nondenaturing and, in some cases, even protein-stabilizing.
Abstract: One of the limitations of immunoaffinity chromatography as been that high-affinity antigen-antibody complexes are difficult to dissociate, often leading to inactivation of the protein product during elution from the immobilized antibody. As described in this unit, some antigen-antibody complexes can be dissociated in the presence of a combination of a low-molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt. These conditions seem to be generally nondenaturing and, in some cases, even protein-stabilizing. This type of antibody is designated "polyol-responsive." These antibodies can be easily identified and isolated as monoclonal antibodies (MAbs) from a typical fusion, using standard hybridoma procedures. They have proven to be very valuable reagents for the immunoaffinity purification of active, labile, multi-subunit protein complexes.
TL;DR: In this unit, protocols are described for hybridizing slide‐mounted paraffin sections or cryosections with labeled probes for In situ hybridization to cellular RNA to determine the cellular localization of specific messages within complex cell populations and tissues.
Abstract: In situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. In this unit, protocols are described for hybridizing slide-mounted paraffin sections or cryosections with labeled probes. Support protocols describe synthesis of 35S-labeled riboprobes and dsDNA probes, which are then detected using film autoradiography or emulsion autoradiography. Another support protocol describes synthesis of digoxigenin-labeled RNA probes, which are non-radioactive and thus have several advantages. They are easily synthesized in large quantities, they are stable for several months, and they can be reused up to three times. An additional advantage of RNA versus DNA probes is that they result in cleaner signals because nonspecifically bound probe is removed during ribonuclease treatment.
TL;DR: Binding-site selection is used to determine the target specificity of a sequence-specific DNA-binding protein in this unit, using a pool of random-sequence oligonucleotides as the source of potential binding sites.
Abstract: Binding-site selection is used to determine the target specificity of a sequence-specific DNA-binding protein. In this unit, a pool of random-sequence oligonucleotides is used as the source of potential binding sites. This pool is incubated with extract containing the DNA-binding protein of interest and the protein-DNA complexes are isolated by immunoprecipitation with an antibody specific for the protein under investigation. Unbound oligonucleotides are removed by gentle washing, and bound oligonucleotides are recovered, amplified by the polymerase chain reaction (PCR), and used as input DNA for a further round of binding, recovery, and amplification. After four rounds of selection, progress of the procedure is monitored by mobility shift analysis of the selected oligonucleotide pools. In the , individual binding sites are isolated from the appropriate complex on a mobility shift gel, cloned into plasmids, and examined by sequencing.
TL;DR: This unit focuses on protocols for generating nested subclones via enzymatic digestion and utilizes Bal 31 nuclease to generate nested deletions for chemical sequencing in conjunction with specialized chemical sequencing vectors.
Abstract: Nested deletions useful for dideoxy DNA sequencing are a set of deletions originating at one end of a target DNA fragment and extending various lengths along the target DNA. Each successively longer deletion brings "new" regions of the target DNA into sequencing range (about 300 bp for normal sequencing gels) of the primer site for a general discussion of nested deletions in DNA sequencing). Two protocols for generating nested subclones via enzymatic digestion are included in this unit. In the first, a set of nested deletions is generated by exonuclease III. The primary advantage of this method is that the deletion products generated from the original clone can be recircularized to generate functional plasmids and thus do not require subcloning into another vector. An alternate method utilizes Bal 31 nuclease to generate the deletions. This method requires subcloning of the deletion fragments into a separate vector for subsequent use. Both methods require the presence of unique restriction sites in the vector that are not present in the insert DNA. Bal 31 can also be used to generate nested deletions for chemical sequencing in conjunction with specialized chemical sequencing vectors.
TL;DR: This protocol is for a cDNA library constructed in the λ>10 vector or its equivalent, which allows a high cloning efficiency and permits elimination of nonrecombinants; however, the protocol can be used to produce subtracted cDNA libraries in any vector system.
Abstract: For some experiments, a complete cDNA library is unnecessary and instead, a subtracted cDNA library is useful. A subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one cell or tissue type and not present in a second type. This cDNA library is used to isolate a set of cDNA clones corresponding to a class of mRNAs, or to aid in the isolation of a cDNA clone corresponding to a particular mRNA where the screening procedure for the cDNA clone is laborious because a specific DNA or antibody probe is unavailable. Since relatively few recombinants are obtained after subtraction, this protocol is for a cDNA library constructed in the lambda>10 vector or its equivalent, which allows a high cloning efficiency and permits elimination of nonrecombinants; however, the protocol can be used to produce subtracted cDNA libraries in any vector system.
TL;DR: A variation on differential display, restriction-mediated differential display (RMDD) presents an alternative approach to the fragment display technologies that touts high sensitivity, a strategy for avoiding false positives, nonradioactive detection, and universal applicability to any polyadenylated RNA.
Abstract: A variation on differential display, restriction-mediated differential display (RMDD) presents an alternative approach to the fragment display technologies. It touts high sensitivity (detection of mRNAs at a dilution of 1:100,000 and of regulation factors lower than two-fold), a strategy for avoiding false positives, nonradioactive detection, and universal applicability to any polyadenylated RNA.