Experimental extraction of λ phage DNA from mass cultures using proteinase K and SDS
DNA can be well isolated from large-scale preparations of λ phage by digestion with an effective protease (e.g., proteinase K) and subsequent phenol:chloroform
Topic
DNA experiment
Techniques for detecting, quantifying and localizing antigens and antibodies — ELISA, Western blotting, immunohistochemistry and flow cytometry. Below are the protocols, FAQs and technical articles in our knowledge base tagged with this topic.
324 resources · protocols, FAQs & articles
Experimental preparation of λ phage particles from large-scale lysates
λ phage prepared from mass cultures can be recovered from the lysate by polyethylene glycolysis (PEG) precipitation in the presence of high salt. The residual
Gel electrophoresis of formaldehyde psyllids
Formaldehyde-agarose gel electrophoresis (formaldehyde-agarose gel electrophoresis) Formaldehyde is a commonly used denaturant for RNA. It is used for (1) RNA i
RAPD (Random Amplified Polymorphic DNA) analysis technique
Polymerase chain reaction (PCR) has greatly influenced almost all areas of molecular biology with its superiority and with its basic procedure and DGGE, a varie
denaturing gradient gel electrophoresis
Denaturing gradient gel electrophoresis (DGGE) is a gel system that enables the separation of DNA fragments according to their melting properties. (1) By settin
In Situ Hybridization Requirements and Procedures
In situ hybridization can be used for (1) solid-phase molecular hybridization and (2) labeled DNA or RNA as probes to detect specific nucleic acid sequences in
Single Nucleotide Polymorphism (SNP) Assay
SNP (Single Nucleotide Polymorphism), or Single Nucleotide Polymorphism, can be used to detect nucleic acid sequence polymorphisms (Polymorphism) due to changes
Oligonucleotide-directed single-stranded DNA mutagenesis experiments
This paper describes a classic protocol consisting of the double primer technique of Zoller and Smith (1984,1987) combined with Kunkel's mutant yield enrichment
Sampling and DNA sequencing
DNA sequences can be enzymatically or chemically degraded to produce a series of sets of DNA fragments that can be resolved by thin-layer denaturing polyacrylam
Experiments on the preparation of denaturing polyacrylamide gels
In sequencing, 4 series of DNA fragments are analyzed by electrophoresis on a thin polyacrylamide gel under denaturing conditions. This protocol describes the p
Inoue method experiments for the preparation and transformation of susceptible Escherichia coli (preparation of super-susceptible cells)
Inoue's method (1990) for the preparation of E. coli receptor cells is good enough to achieve even the transformation efficiency of Hanahan's method (1980). How
Stability screening assay for transfected cells
This experiment is mainly used to obtain eukaryotic cells containing the target exogenous gene.
DNA Sequence Analysis Technology
DNA sequence analysis techniques can be used to (1) analyze the genetic diversity of species, (2) identify new species, and (3) for comparative genomics.
Experimental chemiluminescent detection of digoxin-labeled probes
The digoxin system is over provided as another non-isotopic labeling method, which is detected by coupling an anti-digoxin antibody to one or more fluorescent d
Sieving agarose gel electrophoresis experiments
Sieve agarose gels spread and electrophoresis like regular agarose gels, but they can resolve small fragments of DNA as well as non-denaturing polyacrylamide ge
Purification experiments for mucoid and plasmid clones
The library should be amplified as soon as possible once it is packaged; it can greatly increase the copy number of the library, but there may be some potential
Experiments on the preparation of denaturing templates for sequencing by double deoxy chain termination method
The following method of denaturing plasmid DNA by base denaturation should be used in conjunction with Scheme 3, as sequencing enzymes are used to catalyze the
Double deoxygenation sequencing using E. coli DNA polymerase I Klenow fragments and single-stranded DNA templates
When Sanger and colleagues established the first DNA strand termination extension method, there was only one suitable DNA polymerase available, the Klenow fragm
Dephosphorylation experiments on enzyme section breaks
In the modification of DNA fragments, the dephosphorylation reaction is an important element, which is catalyzed by alkaline phosphatase, an enzyme that removes
Determination of the amount of DNA fixed in ethanol
Determination of DNA content can be used to (1) perform gene fragment ligation experiments. (2) Perform plasmid concentration determination prior to transformat
Experiments with the Hanahan method for the preparation and transformation of susceptible Escherichia coli (efficient transformation strategies)
In the late 1970s and early 1980s, when Dong Hanahan was a graduate student at Cold Spring Harbor Laboratory and Harvard University, he made previously unheard
In vitro mutagenesis using double-stranded DNA as a template: mutant selection experiment with DpnⅠ
This scheme and Scheme 4 use two oligonucleotides and high-fidelity polymerase to guide DNA synthesis using denatured plasmid DNA as a template. In this scheme,
Electroshock transformation experiments in Escherichia coli
Electroshock transformation experiments in E. coli can be used for: easier and faster transformation.
Dideoxy sequencing reaction experiment with Taq DNA polymerase
In addition to its use in PCR reactions, heat-stabilized DNA polymerase can be used as a replacement for sequencing enzymes or Klenow fragments for double deoxy
Radiation autoradiography and DNA sequence reading experiments from sequencing gels
This experiment describes the procedure regarding radioactive autoradiography and the reading of DNA sequences from sequencing gels. This experiment comes from
Sephacryl S-1000 Chromatography for Removal of Small Fragments of Nucleic Acids from Plasmid DNA Preparations
Sephacryl S-1000 chromatography is an alternative method for separating plasmid DNA from small molecules of nucleic acids (DNA and RNA). This method was first u
Multiple sequence alignment and phylogenetic tree construction experiments
Source of content: Guangdong Pharmaceutical University Laboratory Instruction Manual.
In vitro transcription and translation experiments of cloned genes
DNA cloning refers to the process of molecular manipulation in which a DNA fragment containing a target gene or other meaningful DNA is ligated in vitro with a
Purification experiments of phage clones
Phage is a general term for bacteriophages that infect microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. As a type of virus, phages have so
Gene Synthesis Experiment
Gene synthesis can be applied to (1) metabolic pathway synthesis, (2) gene network construction, and (3) vaccine design.
Detection of methylation by sequencing after bisulfite modification
Bisulfite-modified sequencing can be used primarily to detect methylation.
Experiments on stable transfection of mammalian cell genes
Analyzing the function of a gene often requires the formation of a mammalian cell line containing the stably integrated gene. In transfection, approximately 1/1
Gene cloning: efficient receptor cell production
Highly efficient receptor cells can be used for plasmid transformation.DNA cloning refers to the process of molecular manipulation in which a DNA fragment conta
Phage-spreading plate assay for phage spot production
Source of content: compact guide to molecular biology 5th ed.
Preparation of electrolyte gradient gel experiment
Sheen and Seed (1980) created a clever and simple improvement by utilizing different concentrations of electrophoresis buffer at the top and bottom to create an
Oligonucleotide-mediated mutagenesis experiments
The mutated oligonucleotide is used to direct the synthesis of the template, thereby altering the DNA sequence, with a mutation efficiency of 50-80%.
Plate laying and transfer experiments of phage libraries
The library should be amplified as soon as possible once it is packaged; it can greatly increase the copy number of the library, but there may be some potential
Subcloning of target genes
Subcloning of the target gene can be applied to (1) further analysis of the target gene and (2) gene recombination.
Region-specific mutagenesis experiments
Region-specific mutagenesis experiments for (1) gene-induced modification (2) specific expression of genes (3) clinical anti-tumor and other targets.
Plasmid preparation
Plasmid preparation can be used for (1) carrying exogenous genes into bacteria for amplification or expression of the main vector, which has an important role i
Experiments on the preparation of polyacrylamide gels containing formamide
A common problem in DNA sequencing is the compression of electrophoretic bands due to the presence of DNA dipolar structures. A simple and straightforward solut
Ligation, transformation and screening of recombinant plasmids
Recombinant plasmid ligation, transformation and screening are mainly used to obtain recombinants containing the target gene linkage.
Experimenting with connectors on protruding ends
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Plate-laying and transfer experiments for stickies and plasmid libraries
The library should be amplified as soon as possible once it is packaged; it can greatly increase the copy number of the library, but there may be some potential
Uracil-containing single-stranded phage M13 DNA preparation experiments
The classical Kunfcel oligonucleotide-directed mutagenesis method utilizes the selective action of uracil DNA glycosylase in E. coli to specifically screen out
merged oligonucleotide mutagenesis assay
An important feature of this method is the direct cloning of single-stranded, abbreviated oligonucleotides into homologous double-stranded molecules after their
Targeted cloning experiments in plasmid vectors
Most commonly used plasmids contain multiple cloning sites that can be recognized by different endonucleases. Because of the large number of cloning sites avail
Amplification experiments for stickies and plasmid libraries
The library should be amplified as soon as possible once it is packaged; it can greatly increase the copy number of the library, but there may be some potential
Pulsed field gel electrophoresis experiment
Pulsed-field gel electrophoresis, which alternates periodically between two differently oriented electric fields, allows the separation of DNA molecules up to 5
Amplification experiments of phage libraries
The library should be amplified as soon as possible once it is packaged; it can greatly increase the copy number of the library, but there may be some potential
Experimental double deoxy sequencing reaction with T7 phage DNA polymerase
This protocol is modeled after the method of Tabor and Richardson (1987a, 1989b, 1990), which uses a single-stranded DNA template for DNA sequencing (for single
Expression experiments of multidrug resistance genes
Expression experiments of multidrug resistance genes are mainly used for tumor therapy research.
Chemical sequencing assay experiment
The importance of chemical sequencing has always been demonstrated by obtaining sequences of oligonucleotides, functional analysis of transcriptional regulatory
Cycle sequencing: double deoxy sequencing experiments using PCR and primer end-labeling
The key to the success of this method is the purity of the template DNA. Contamination with agarose, salt, and protein can lead to premature termination or susp
P1 Transfer of phage clones between E. coli hosts
The yield and quality of P1 phage DNA obtained from different E. coli strains depends on the genotype of the host bacterium and the specific sequence of the exo
Amplification and storage of mucoid libraries (amplification in liquid medium)
Do not over-amplify the mucoid library, as this will inevitably cause distortion of the original genome. Faster-growing clones will be over-presented, unstable
Screening of unamplified mucoid libraries by hybridization (filter film photocopying)
Thick bacterial colonies transformed by mucilage can be screened by hybridization, a method derived from the large-scale screening of plasmid-transformed coloni
Experiments with tetracycline as a regulator of inducible gene expression
The following protocol is divided into 3 stages: stable transfection of fibroblasts with pTet-tTAk, stable transfection of NIH-3T3 cells inducible to express tT
Conjugate transcription profiling experiments
Nuclear contig analysis is used to determine whether a gene is transcriptionally regulated in a given clone. Theoretically, nuclear contig analysis should be pe
Gel blocking assay of DNA binding proteins
The gel-blocking assay for analyzing DNA-binding proteins was invented by Fried and Crothers (1981) and was the first to provide a dynamic method for analyzing
Mapping of protein binding sites on DNA by DNAase I footprinting assay
This protocol describes a mapping method for identifying protein binding sites on radiolabeled DNA fragments. The protocol uses DNAase I to cut the DNA, whereas
Rapid analysis of λ phage isolates (purification of λ DNA from plate lysates) assay
Analysis of promising genomic or cDNA clones usually begins with digestion of small preparations of λ phage DNA with restriction enzymes and analysis of the di
DNA transfection experiment using Polybrene
The Polybrene and DMSO transfection method described below is a modification of the method of Aubin et al. (1997). Using this method, plasmid DNA can be efficie
Amplification experiments of genomic libraries
Recombinant phage libraries can be amplified by plate culture, and the original seed for plate culture can be derived directly from the packaging mixture descri
Bioparticle-mediated DNA transfection assay
The following scheme is based on methods developed by Horch et al. (1999), Sanford et al. (1993), publications from major gene gun manufacturers (US/EG Bulletin
DEAE-dextran-mediated high-efficiency transfection assay
Two forms of the classical DEAE-dextran transfection method are introduced here. The first one (the main option), in which the cells are exposed to high concent
Lipofection-mediated DNA transfection assay
The following method is a modification of one studied by Mark Evans (Alexion Pharmaceuticals, New Haven, Connecticut). This experiment was derived from the next
Experiments on the purification of proteins with histidine tags by solidified Ni2+ absorption chromatography
Polyhistidine binds to a variety of transition metals and transition metal chelates, so proteins with exposed His-6 bind to cured Ni2+ resins, and target protei
Expression of cloned genes in Escherichia coli using the T7 phage promoter experiments
Tabor and Richardson in 1985 and Studier and Moffatt in 1986 proposed a new expression system utilizing the T7 phage promoter, using transcriptional signals obt
Expression of clonized genes in Escherichia coli using IPTG inducible promoter experiment
This protocol will discuss the factors affecting plasmid expression efficiency in the Troubleshooting and Optimization of Inducible Promoter Expression Proteins
Selection of plasmid vectors to construct expression libraries
There are three methods for detecting bound antibodies: radiochemical screening, colorimetric screening, and chemiluminescent screening, the advantages and disa
Single-strand conformational polymorphism and heteroduplex analysis methods for mutation detection experiments
The purpose of sinister mutation testing is mainly for figuring out the molecular mechanisms of human hereditary diseases in order to effectively target these d
Screening experiments of intermediate cell clones by hybridization method
This method is applicable to bacterial clones of any size, but small clones give the clearest results. Small clones produce sharp hybridization signals and are
Screening experiments of small quantities of bacterial clones by hybridization method
Bacterial clones on the main agar plate and on a nitrocellulose filter membrane or nylon membrane covering the surface of another agar plate can be localized, a
Overlapping extensions generate site-specific mutagenesis experiments
Four primers are required for site-specific mutagenesis by the overlapping extension method (see Fig. 13-4) (Higuchi et al. 1988, Hetal. 1989). This experiment
Efficient and rapid sentinel mutagenesis in the same tube using large-primer PCR
The large primer method was originally established by KAMMANN et al. (1989) and the current method has been modified by many researchers including Sarkat and So
USE mutation experiment
In this method, two oligonucleotide primers hybridize to the same strand of the DNA duplex of a denatured recombinant plasmid. One primer (mutagenic primer) car
Determination of chloramphenicol acyltransferase content by thin-layer chromatography experiment
In this protocol, we list three different assays that can be used to determine CATase activity expressed after transfection of mammalian cells with the reporter
Calcium phosphate-mediated plasmid DNA transfection in eukaryotic cells
The calcium phosphate-mediated plasmid DNA transfection of adherent cells described in this method is an improvement on the method established by Jordan et al.
Lysis experiments with bacterial clones
Lysis experiments with bacterial clones can be used to (1) construct a phage lysogen from a recombinant phage expressing a specific fusion protein, and (2) indu
Amplification and storage of mucoid libraries (amplification on filter membranes)
Applying this amplification method, the library is not prone to distortion, due to the fact that mixed colonies containing different recombinant mucilages do no
Calcium phosphate-mediated transfection of high molecular weight genomic DNA in cells
The following method is a modification of the phosphotransformation method established by Graham and Van der Eb (1973), in which high molecular weight genomic D
Determination of luciferase in mammalian cell extracts
The assay of luciferase in mammalian cell extracts can be used to (1) measure the amount of luciferase in animal tissues, and (2) measure other enzymatic activi
Experimental purification of fusion proteins by glutathione agarose affinity chromatography
The exogenous protein expressed in the pGEX vector is fused to glutathione S transferase and can therefore be purified by glutathione-agarose affinity chromatog
DNAase I hypersensitive site mapping
DNAase I hypersensitive site mapping is frequently used to localize regulatory regions of eukaryotic genes. For reasons that are not yet understood, genes carry
Purification of expressed proteins from inclusion bodies
This experiment was mainly used to obtain soluble active proteins.
Exonuclease III digestion produces multiple sets of nested deletion mutants
The nested deletion mutagenesis method, in which multiple oligonucleotides are progressively deleted from one or the other end of the target DNA, has been used
Screening of fixed-point mutagenesis recombinant cloning experiments by hybridization with radiolabeled oligonucleotides
This protocol mainly describes the screening of recombinant phage M13 clones, whereas one option is to screen bacterial clones containing phage particles. Final
Cross-reactive antibody removal assay in antisera
This protocol describes a method for removing antibodies reactive with bacterial-encoded proteins from polyclonal antisera by co-warming the antiserum with bact
Screening experiments of large numbers of bacterial clones by hybridization methods
This method was reported by Hanahan and Meselson (1980, 1983) and is primarily used to treat bacteria transformed by plasmid cDNA libraries. A mixture of transf
BAL31 Nuclease digestion assay for generating bidirectional deletion mutants experimental
This protocol uses the nuclease BAL31 (purified from the marine bacterium Alteromonas espejiana BAL31) to produce unidirectional or bidirectional deletions in c
Experimental preparation of λ phage particles from large-scale lysates
λ phage prepared from mass cultures can be recovered from the lysate by polyethylene glycolysis (PEG) precipitation in the presence of high salt. The residual
Antibody assay for removal of cross-reactivity in antisera
The methods described in this section are only for the preparation of antisera containing relatively low-titer anti-E. coli antibodies. If the treated antiserum
Affinity chromatography assay for removal of cross-reactive antibodies
This section describes a method for removing components of crude immunoglobulin that react with bacterial-encoded proteins using a matrix that binds bacterial p
Lysogenic infection assay on agar plates
Fusion proteins encoded by λ phage recombinants can be prepared by lysogenizing colonies of E. coli strain Y1089. However, preparation of lysogenic bacteria fr
Experimental purification of λ phage particles by centrifugation in a graded gradient of glycerol
This protocol is suitable for the preparation of phage progenitors, the preparation of phage particles (used to obtain DNA for subcloning), and the preparation
Experimental extraction of λ phage DNA from mass cultures using proteinase K and SDS
DNA can be well isolated from large-scale preparations of λ phage by digestion with an effective protease (e.g., proteinase K) and subsequent phenol:chloroform
Lysogenic infection assay in liquid cultures
This method is suitable for rapid screening of immunodetectable fusion proteins against λgtll recombinant phage. However, under other conditions (e.g., optimiz
Experiments on expression of cloned genes in E. coli using λ phage PL promoter
The λ phage PL promoter is a potent promoter controlled by a temperature-sensitive repressor (cIts857), which can repress transcription of the PL promoter at l
Experimental purification of maltose-binding fusion proteins by affinity chromatography with straight-chain starch resin
Affinity chromatography methods can virtually purify maltose-binding fusion proteins into single components. Maltose-binding protein self (MBP) is a periplasmic
Experiments on secretion and expression of exogenous proteins with alkaline phosphatase promoter and signaling sequences
This experiment describes the process of expressing exogenous proteins in Escherichia coli using the alkaline phosphatase promoter (phoA) and signaling sequence
Preparation of radiolabeled DNA probes using the polymerase chain reaction
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Labeling of DNA by random primer method
Using oligonucleotides as primers, DNA polymerase can initiate DNA synthesis along a single-stranded template (Gotilian 1969). If the sequences of the oligonucl
Application of mixed oligonucleotide primer-guided cDNA amplification (MOPAC)
The best way to clone a protein for which only part of the sequence is known is to design an oligonucleotide using the known amino acid sequence and use this ol
Rapid amplification of cDNA 5' ends (5'-RACE)
There is no greater failure in molecular cloning than the isolation of a cDNA clone that lacks the characteristic structure of the sequence at the 5' end of the
polymerase chain reaction (PCR)
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Analysis of RNA by primer extension method
Primer extension is primarily used for mRNA 5' end mapping. poly (A) + RNA is first hybridized to an excess of a 5' end-labeled single-stranded oligonucleotide
Screening poly (A)+ RNA by batch chromatographic methods
When working with many RNA samples or working with small amounts of total mammalian RNA (<50 μg), a batch chromatography method with oligo (dT)-cellulose is
Extraction of poly(A) + RNA by oligo(dT)-cellulose chromatography
In contrast to rRNA, 5SRNA, 5.8 SRNA and tRNA, most eukaryotic mRNAs have a poly(A) tail at their 3' end. Therefore, mRNA can be isolated from total cellular RN
Purification of RNA from tissues and cells by acid phenol-guanidine thiocyanate-chloroform extraction
The cellular RNAase should be inactivated as soon as possible during the first stage of the extraction process. Once the endogenous RNAase is destroyed, the pos
Southern blotting (DNA transfer from one agarose gel to two membranes simultaneously)
DNA can be transferred from both sides of the agarose to both membranes simultaneously. This method is useful when the same restriction endonuclease fragment ne
Pulsed-field gel electrophoresis with transverse alternating fields
Gardiner et al. (1986; Gardiner and Patterson 1989) used a gel electrophoresis device with two sets of platinum wire electrodes placed on either side of a verti
Molecular quality criteria for pulsed-field gel electrophoresis
PFGE is used to isolate very large DNA molecules and thus requires standards of very high molecular mass. Such standards can be obtained from phages such as T7
Neutral polyacrylamide gel electrophoresis
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Recovery of DNA from agarose and polyacrylamide gels (electroelution to dialysis bags)
This technique (McDonell et al., 1977) allows the recovery of double-stranded DNA from agarose or polyacrylamide gel slices over a wide range of molecular masse
Direct cDNA screening by large fragment genomic DNA cloning
This protocol uses a 500kb contiguous sequence of human genomic DNA cloned in a BAC vector to directly screen for cDNAs complementary to it, but the method can
Exon capture and amplification
This protocol describes the method of exon amplification using the mammalian shuttle vector pSPL3 as an example, which is divided into the following five stages
Eukaryotic expression library construction and screening experiments
Similar to Scheme 1, Scheme 2 describes the method of cDNA library construction and screening on eukaryotic expression vectors, and the process is divided into
Hybridization experiments with oligonucleotide probes in solution
Jabobs et al. (1988) described the method and principles of hybridization in quaternary ammonium-containing buffers. The following is a simple variation of this
Experiments with oligonucleotides synthesized by labeling with Escherichia coli Klenow fragments
Oligonucleotides are usually labeled using a phosphorylation reaction catalyzed by the T4 phage polynucleotide kinase. However, there is sometimes a need to lab
Experiments on purification of radiolabeled oligonucleotides by Sep-Pak C18 column chromatography
This scheme describes a method to separate radiolabeled oligonucleotides from undoped radiolabel using the property of antiphase affinity of oligonucleotides fo
Polyacrylamide gel electrophoresis purification of oligonucleotide experiment
The method detailed in this protocol for the purification of crude synthesized oligonucleotides using denaturing polyacrylamide gel electrophoresis is a modific
Application of yeast artificial chromosomes
Until the mid-1980s, there were no techniques for isolating, amplifying, and analyzing large sticky fragments of genomic DNA. Prior to that time, if a DNA fragm
Isolation of BAC DNA from bulk cultures
The amount of DNA required for fine analysis of recombinant BAC, including detailed restriction mapping, DNA sequencing or subcloning, is greater than can be pr
Isolation of BAC DNA from small cultures
Small amounts of BAC DNA were prepared from 5 ml of BAC-transformed cell cultures.The DNA was prepared by alkaline lysis.The yield of BAC DNA can be as high as
Application of bacterial artificial chromosomes
The bacterial artificial chromosome (RAC ) is a synthetic vector based on the E. coli fertility factor (F). This experiment is based on the "Guide to Molecular
GFP and fluorescence resonance energy transfer techniques for protein interaction assays
We have divided the protocol into three phases: the first phase describes the preparation of proteins and the labeling of proteins with fluorescent dyes; the se
Immunoprecipitation assay to determine binding proteins
Many of the protein-protein interactions present in intact cells are maintained when cells are lysed under nondenaturing conditions. This rhopper can be used to
GST fusion protein sedimentation assay
The GST sedimentation experiment has an advantage over far western (Scheme 2): the probe protein is incubated with a possible partner protein in a more natural
Experiments with two-hybrid and other two-component systems
This experiment describes experimental methods regarding two-hybrid and other two-component systems. This experiment comes from the next volume of the Laborator
Ecdysone regulation induces cellular gene expression experiments
The following protocol is modified from the protocol that accompanies the Ecdysone-Induced Mammalian Expression System (Invitrogen); a similar system is sold by
Alkaline agarose gel electrophoresis
Alkaline agarose gel electrophoresis is performed at high pH, which causes the loss of a proton from thymine and guanine residues, thus preventing the formation
Experiments on purification of radiolabeled oligonucleotides by ethanol precipitation method
If the radiolabeled oligonucleotide is to be used only as a hybridization probe, it is generally not necessary to completely remove the unadulterated radiolabel
Protein-protein interaction assay with GST fusion proteins
GST fusion proteins, as recombinant proteins expressed in bacteria, have been used in thousands of research projects since their introduction (Smith and johnson
Experimental Determination of Unwinding Temperature
The unlinking temperature for hybridization of oligonucleotides to target sequences can be calculated using the method described in the overview of this chapter
Experimental purification of radiolabeled oligonucleotides by size exclusion chromatography
When radiolabeled oligonucleotides are used in enzymatic reactions such as primer extension reactions, it is necessary to completely remove the unadulterated ra
Genetic engineering with PCR
This protocol (provided by Andereson of the University of Texas Southwestern Medical Center) describes the introduction of restriction endonuclease sites at bot
Pulsed-field gel electrophoresis with clamped uniform electric field
In clamped homogeneous electric field (CHEF) gel electrophoresis, the electric field is generated by multiple electrodes. These electrodes are arranged in a qua
Analysis of interacting proteins using BIAcore
BIAcore surface plasmon resonance is widely used for (1) studying the interaction process between various biomolecules (e.g., peptides, proteins, oligonucleotid
Recovery of DNA from agarose gels (DEAE-cellulose membrane electrophoresis)
Recovery of DNA from agarose gels can be applied to (1) mammalian cell transformation and (2) radiolabeling.
Rapid isolation of mammalian DNA
Mammalian DNA prepared according to this protocol is approximately 20-50 kb and is suitable for use as a template for PCR reactions. the DNA yield varies betwee
Random priming method (radiolabeling of DNA using random oligonucleotide extensions in the presence of melted agarose)
This method can be used for radiolabeling of DNA recovered from low melting point agarose gels (Feinberg and Vogelstein 1983, 1984). This experiment is based on
Introduction of restriction endonuclease sites at the end of amplified DNA products by PCR amplification
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Ribonuclease protection (mapping of RNA with ribonuclease and radiolabeled RNA probes)
Ribonuclease protection analysis is used to measure the abundance of specific mRNAs and to map their topological heterogeneity. This method involves hybridizati
Application of mRNA reverse transcription amplification of cDNA (RT-PCR)
RT-PCR can be used to: (1) construct large-capacity cDNA libraries; (2) identify mutations and polymorphisms in transcribed sequences; (3) measure the intensity
DNA Agarose Nucleic Acid Electrophoresis
Nucleic acid electrophoresis is an important tool for conducting nucleic acid research and can be used for techniques such as (1) nucleic acid probing; (2) nucl
DNA blotting and narrow line blotting experiments
Spot and narrow line blotting experiments that can be used to detect the relative abundance of target sequences in blotted 1DNA products. The main applications
Experiments on DNA precipitation
The experiment of precipitating DNA can be applied to isolate DNA.
M13 Experiments on the preparation of phage-derived vectors
M13 phage is a filamentous phage that does not lyse bacterial cells after infection, but only utilizes intracellular material to complete its own proliferation,
cDNA library construction
The cDNA library construction can be: (1) used to isolate full-length genes for gene function studies; (2) screen target genes and use them directly for express
Labeling experiments with DNA probes
Nucleic acid probe molecular hybridization refers to the two nucleic acid single strands with certain homology can be formed under certain conditions according
Bacterial Genome DNA Extraction
Bacterial genomic DNA extraction can be applied to: (1) obtain bacterial genomic DNA; (2) as PCR template; (3) for sequencing, genetic information analysis, etc
Chromosome CBG specimen preparation experiment
Heterochromatin is condensed throughout interphase and metaphase and is therefore relatively constant in size. They are usually located near the mitotic grains
Formaldehyde denaturation electrophoresis of RNA
Formaldehyde denaturation electrophoresis of RNA can be used for: (1) after extracting the total RNA of the sample, the quality of RNA is generally judged accor
Animal DNA Extraction
Animal liver DNA extraction can be (1) used for forensic identification, (2) for diagnostic and phylogenetic studies, and (3) for further polymerase chain react
BSP Clone Sequencing
Methylation testing of DNA
Rapid amplification of cDNA ends
Rapid amplification of cDNA ends
Basic experiments of FISH technique
Source : Molecular Cytogenetics Techniques and Applications
Notch translation experiment
Source : Molecular Cytogenetics Techniques and Applications
Poxvirus purification assay
Poxvirus is usually purified by sucrose density gradient centrifugation. The purified virus can be used to prepare poxvirus DNA for use in studies that do not a
PCR-based differential subtractive cDNA cloning experiment
Differential cloning is an effective technique for isolating and characterizing mRNAs that are differentially expressed in two cell populations. The cell types
Differential reduction cDNA library creation experiment
Differential subtraction libraries contain mRNA cDNA clones that are present in one cell or tissue but not in another. This cDNA library is used to isolate a se
Hybridization and data processing experiments
By determining the ratio of fluorescence at each immobilized cDNA spot, one can measure the relative amount of messengers in one sample library to another. If a
Fluorescence immunophenotyping and FISH co-analysis experiments on tumor cells
Chromosomal aberrations are usually detected in most hematological and various solid tumors and, clinicopathologically, are frequently associated with the deter
FISH assay for detection of HER2 amplification in breast cancer
Gene amplification is frequently detected in human tumor cells and plays an important role in tumorigenesis. Genome-wide scans of tumor cells using comparative
Chromogenic in situ hybridization and FISH in pathology
Fluorescence in situ hybridization (FISH) techniques enable rapid detection of chromosomal abnormalities in a variety of tissues, including fresh and archival s
Experiments in primer-mediated in situ labeling techniques
Primed in situ labeling (PRINS) is a hybridization technique that stains cells and tissues for specific DNA sequences, and it is capable of obtaining the same t
Chromosome Microdissection Experiment
It has been well known in recent decades that chromosomal rearrangements are present in most human tumors and in certain specific human genetic diseases. Tumor-
Splenic necrosis virus-based vectors
The experimental approach described in this chapter is based on our experiments with carriers from S N V and its closely related R E V -A (P a r v e e n e t a L
BAC/PAC library construction
BAC (Bacterial Artificial Chromosome) libraries can be used for (1) whole genome sequencing; (2) construction of physical maps, chromosome step-by-step; (3) gen
Intracellular injection of mRNA
Modern Neuroscience Research Techniques Author(s): U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun
Construction and screening of cDNA libraries-Experimentation of methods for identification of newly expressed genes under external environmental stress conditions
Materials Many organisms are excellent models for disease studies or for exploring the molecular adaptive mechanisms that enable cells and organisms to cope wit
Series of experiments for analyzing gene expression
SAGE (Velculescuetal.1995) is a highly sensitive method based on PCR technology to study gene expression and to obtain qualitative and quantitative information
Differential Display Technology (DD) Experiment
D D was first proposed by Liang and Pardee in 1992. To date, about 1700 articles related to DD have been published, which shows its great influence. Many genes
Labeling the 3' protruding end of double-stranded DNA with [α-32P] 3'deoxyadenosine 5'-triphosphate or [α-32P] dideoxyATP ends
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Labeling the 3' end of double-stranded DNA with the Klenow fragment of E. coli DNA polymerase I
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Combined with magnetic bead experiments
This step is only part of Experiment A in the cDNA synthesis for serial analysis of gene expression. In Experiment B, the cDNA is already bound to the streptavi
Construction of DNA libraries and their screening
The construction and screening of DNA libraries can be used to: (1) cut the entire genomic DNA of an organism into DNA fragments of a certain length by restrict
Experiments on the application of the comet assay to environmental toxicology
The comet assay, also known as the single-cell gel electrophoresis assay, is a method used to detect D N A damage in almost all nucleated cells. The comet assay
ULS Labeling Experiment
Source: Molecular Cytogenetic Technology and Applications
Plasmid Extraction
Purification of plasmid DNA from bacteria
Metabolic labeling experiments at isolated synapses
mRNA and rRNA are present within dendrites and axons (VanMinnen 1994; Steward 1997). It is puzzling whether mRNAs located in regions outside the cytosol are act
Chromosome GTG specimen preparation experiment
Non-banded chromosomes are difficult to identify except for some chromosomes based on their morphology, whereas the banding technique allows the appearance of d
Experiments on in situ hybridization techniques for cultured neurons
The purpose of in situ hybridization (ISH) is to visualize TKmRNA molecules within cellular structures. High temperatures are used in the ISH procedure, which p
Duplex Oligonucleotide Primer PCR (DOP-PCR)
Source : Molecular Cytogenetics Techniques and Applications
Color Tape Experiment
The interspecies color banding technique is a simple and rapid method for detecting chromosomal abnormalities that cannot be identified by G-banding. This techn
Electron microscope in situ hybridization technique experiment
Electron microscopy in situ hybridization experiments can be used to (1) study the subcellular localization of specific nucleotide sequences; (2) many researche
Extraction of plasmid DNA from Escherichia coli (alkaline lysis)
The basic principle of plasmid DNA extraction by alkaline lysis is that solution I suspends the cells and EDTA can chelate with Ca2+ and Mg2+ to inhibit the act
Difluorokinase assay
Luciferase plasmids were constructed and then transfected into cells to detect alterations in reporter gene expression by comparing overexpression or interferen
Genomic DNA preparation in mammals
Various factors that break and degrade DNA should be avoided as much as possible during DNA extraction to ensure the integrity of DNA and lay the foundation for
Multicolor fluorescence in situ hybridization assay
M-FISH has been successfully used to identify marker or derived chromosomes in congenital diseases. Recently, this method has been increasingly used to identify
Interphase FISH study experiments in chronic myeloid leukemia
The purpose of this chapter is to apply fluorescence in situ hybridization to the study of interphase nuclei in patients with chronic myeloid leukemia (CML). In
PCR products electrophoretic silver staining assay
There are two commonly used nucleic acid electrophoresis methods. ① Horizontal electrophoresis: agarose gel, bands are judged under UV light. This method is ec
FISH Basic Techniques and Problem Solving
Fluorescence in situ hybridization (FISH) technology allows the detection of specific nucleic acid sequences in chromosomes, cells and tissues. The unambiguous
λ Purification of phage arms (by sucrose density gradient centrifugation) experiments
Unlike insertion vectors, replacement vectors contain a filler fragment in the middle of the genome, which must be removed in order to accommodate the foreign D
Partial cleavage (pre-reaction) of eukaryotic DNA for genomic libraries
Regardless of the base composition and sequence of high-molecular-quality DNA, it can be fragmented in a semi-random fashion by hydrodynamic shearing. However,
Extraction of λ phage DNA from large-scale cultures with formamide
Instead of SDS/proteinase K, formamide can be used to remove the shell from purified phage particles. This method is not very efficient, but in a sense it is fa
Preparation of λ phage DNA that can be cleaved by a single restriction enzyme and used as a cloning vector
In some cases, prepared λ phage DNA can be used for cloning after simple digestion with restriction enzymes. However, this option is only feasible if the
Preparation of λ phage DNA by double restriction enzyme cleavage as a cloning vector
Replacement vectors (e.g., λ2001, λDASH, EMBL series, Charon 34, Charon 35, and Charon 40) contain a series of restriction sites that are aligned
Determination of DNA content in λ phage progeny and lysates by gel electrophoresis experiment
This program describes a method for rapid estimation of the DNA content in λ phage protospecies. The method can be used firstly to find out whether the y
Lysis of colonies and DNA binding assay to filter membranes
This protocol describes how to release DNA from a clone carrying a recombinant plasmid and immobilize it on a nitrocellulose filter or nylon membrane, a method
λ Mass culture (low multiplicity infection) experiments with phages
Mass preparation of λ phage can be obtained by either low-mass infection of bacterial cultures or high-mass infection. Immediately after low magnificatio
Preparation of λ phage proto-species experiments with small quantities of liquid cultures
High-titer λ phage progenitors can be prepared from small quantities of liquid cultures. This method was first used by Leder et al. (1977). In general, i
Preparation of λ phage proto-species assay by plate lysis and elution
The preparation of λ phage progenitors derived from a single phage spot is usually done by two methods: (i) plate lysis, which is a method of phage proli
Hybridization of bacterial DNA on filter membranes
This protocol describes how to hybridize radiolabeled probes to DNA of transforming bacteria immobilized on filter membranes, and methods for recovering clones
λ Phage phage spot picking experiments
Genetically homologous λ phage protospecies are obtained by "picking" a completely separate phage spot and preserving agar/agarose containing the lysate
λ Experiments on spread plate culture of phages
Phages originate from the infection of a single bacterium by a single phage particle. The daughter virus particles synthesized after the first infection adsorb
Screening of bacterial colonies with X-gal and IPTG (alpha complementary)
The chromogenic substrate X-gal can be mixed with bacterial cultures, mixed with solubilized top agar and spread on selective culture plates. This experiment is
Enzymatic digestion of DNA fragments
Restriction endonucleases are a class of DNA hydrolases that recognize specific nucleotide sequences in double-stranded DNA molecules, and the discovery and app
Total DNA quality and digestion assay
The objectives of the experiment are to understand and master the methods of detecting the quality of DNA as well as the methods of DNA quantification; to under
Rapid small volume extraction of total plant DNA
DNA molecule is the basic material of molecular biology research, according to different experimental purposes can be taken to different extraction methods to o
Ligation of plasmid and target DNA in low melting point agarose experiment
The most time-consuming step of the plasmid cloning operation is the purification of the desired sized fragments of exogenous and plasmid DNA by electrophoresis
Toward Flat-Ended DNA Ligation Synthesis Junction Experiment
A junction is a synthesized DNA fragment that is complementary to itself, usually 8-16 nucleotides in length, and the complementary fragment can be complexed to
Establishment of randomized overlapping DNA insertion libraries
The following protocol describes the starting steps of birdshot sequencing, from shearing of target DNA to construction of a DNA fragment library with M13 phage
Dephosphorylation of plasmid DNA
Removal of the phosphate group at the 5' end prevents self-ligation and cyclization of plasmid DNA. In an in vitro ligation reaction, DNA ligase forms a phospho
Removal of small fragments of nucleic acids from plasmid DNA preparations by lithium chloride precipitation
The principle of lithium chloride precipitation for removing small fragments of nucleic acids (both DNA and RNA) from plasmid preparations is based on the fact
Removal of small fragments of nucleic acids from plasmid DNA preparations by NaCl centrifugation
Conventional plasmid DNA preparations are contaminated, to varying degrees, with small molecules of DNA or RNA from the genome of the host bacterium or plasmid
Removal of ethidium bromide from DNA by ion exchange chromatography
Ethidium bromide in DNA purified by cesium chloride gradient centrifugation can be removed by ion-exchange chromatography, followed by ethanol precipitation to
Removal of ethidium bromide from DNA by extraction with organic solvents
To remove ethidium bromide from CsCl-gradient purified DNA, repeated extraction with organic solvents is commonly used. Subsequently, CsCl can be removed by dia
Plasmid DNA purification by polyethylene glycol precipitation assay
This method was first devised by Richaed Treisman (ICRF, London, UK) with reference to the work of Lis, who was the first to use polyethylene glycol (PEG) to is
Chemiluminescent Dideoxy DNA Sequencing Assay
Standard double deoxy DNA sequencing can be easily and efficiently detected using non-isobaric labeled chemiluminescence. In the sequencing reaction, biotin-lab
Junction partitioning mutagenesis of DNA
Linker scanner mutations: In vitro additions of sites to restriction fragments that result in recombination of two DNA molecules, resulting in the addition of a
Freeze-thaw method for recovery of DNA fragments
The freeze-thaw method of recovering DNA fragments can be used to (1) obtain purified DNA fragments and (2) recover PCR products.
Steps for SSR labeling experiments
SSR simple sequence repeat markers can be used to: design pairs of primers with a specific sequence of microsatellite regions, and polymorphism of SSR loci amon
Gene transfection (calcium phosphate-DNA co-precipitation)
Gene transfection technology can be applied to: genome function research (gene expression regulation, gene function, signal transduction and drug screening rese
Construction of recombinant DNA by polymerase chain reaction
The polymerase chain reaction constructs recombinant DNA for a wide range of uses (1) for sequencing primers (2) targeted mutagenesis (3) nucleic acid hybridiza
DNA blot hybridization analysis experiment
Hybridization analysis is based on the principle that a single-stranded DNA molecule of a specific sequence (i.e., a commonly labeled "probe") can form a base p
Agarose gel electrophoresis of DNA
DNA electrophoresis can be used to (1) separate DNA fragments of different sizes, (2) identify target DNA fragments, and (3) purify and recover DNA fragments.
Screening of expression libraries constructed in λ phage vectors
In typical immunoscreening experiments, libraries of λ phage expression vector constructs should be spread on plates of E. coli strains that do not conta
Plasmid DNA preparation by the toothpick method in small quantities
Colonies picked with a toothpick from agar medium can be used directly to prepare plasmid DNA. the resulting closed-loop DNA often has too many impurities to be
Transfection of eukaryotic cells using DEAE-glucan
Mouse L-type fibroblasts were used to perform the conditions eubiquitously, but can be applied to almost any cell with only minor modifications. It is critical
DNA preparation of salmon sperm stretcher
Using this scheme, well-sheared, high-quality salmon sperm stretcher DNA can be obtained, which can be used in transformation experiments and other experiments
DNA binding protein assay
This experiment was used to detect sequence-specific DNA-binding proteins in crude extracts.
T4 DNA polymerase assay
T4 DNA polymerase has a human template-dependent polymerase activity, as well as exonuclease activity for the 3' to 5' ends of single- and double-stranded DNA,
Experimental purification of λ phage particles by isodensity gradient centrifugation with CsCl
CsCl iso-density gradient centrifugation is used to prepare the highest purity infectious λ phage pellet, which is free of any bacterial nucleic acid con
Preparation of cytoplasmic S-100 components
The contents of a cell contained within the cell membrane. In eukaryotic cells it refers to the portion of the cell membrane outside the inner core that contain
Screening of DNA binding proteins using λ phage libraries
Synthetic double-stranded oligonucleotides can be used to screen cDNA expression libraries constructed in λ phage to identify clones corresponding to spe
Experiments on preparation of DNA probes labeled with potenticin
The digoxin system is over provided as another non-isotopic labeling method, which is detected by coupling an anti-digoxin antibody to one or more fluorescent d
Analysis of radioisotope-labeled DNA sequence determination
Radioisotope-labeled DNA sequencing analysis can be applied to analyze gene structure-function relationships.
Determination of DNA by chemical method - Diphenylamine colorimetric method
Chemical determination of DNA content can be used for (1) estimation of plasmid concentration prior to transformation and transfection, and (2) assay of acquire
λ Alkaline phosphatase treatment assay of phage vector DNA
Removal of the terminal 5'-phosphate groups from the inner ends of the two arms of the λ phage effectively prevents self-association and reduces the back
Recovery of DNA from low melting point agarose gels (digestion with agarase)
DNA can be recovered from low melting point agarose gels by agarase digestion (Bumeister and lehrach 1989). In this method, the agarase enzyme hydrolyzes the ag
Radiation autoradiography of DNA in polyacrylamide gels
Radioactive DNA bands separated by acrylamide gel electrophoresis can be detected by radioautography. Polyacrylamide analytical gels containing radioactive DNA
DNA preparation by pulsed-field gel electrophoresis (isolation of mammalian cell and tissue DNA)
This protocol describes methods for preparing DNA samples from cultured cells and tissues. Leukocytes of patient or animal origin can also be used as high molec
DNA preparation by pulsed-field gel electrophoresis (isolation of yeast intact DNA)
To prepare yeast DNA for electrophoresis, the cell walls of yeast cells are enzymatically disrupted and then suspended in melted low melting point agarose to ma
Oligonucleotides and excess dNTP were removed from DNA amplification products by ultrafiltration.
At the end of the PCR reaction, it is essential to remove oligonucleotide primers, primer dimers and dNTP from the amplified DNA product. This experiment is bas
Restriction endonuclease digestion of DNA in agarose gel plugs
Chromosomal DNA isolated from mammalian cells, yeast or bacteria can be digested in agarose plugs with the desired endonuclease. The low melting point agarose p
Southern hybridization of radiolabeled probes with membrane-immobilized nucleic acids
The signal intensity obtained by Southern hybridization depends on several factors, including the ratio of immobilized DNA complementary to the probe, the size
RNA mapping with S1 nuclease
Three different nucleases, S1 nuclease, RNAase, and exonuclease VII, are used to quantify RNA, to identify intron positions, and to characterize the positions o
Dot hybridization and narrow line hybridization of purified RNA
The dot and narrow-line hybridization technique (Kafatos et al. 1979) is used to immobilize several nucleic acid samples on the same solid-phase support (usuall
Northern hybridization
RNA samples transferred and immobilized to membranes can be hybridized to specific probes that can be used to localize the RNA of interest. Depending on the exp
Transfer and immobilization of denatured RNA on membranes
In most cases, to detect a specific target mRNA, the RNA is separated by agarose electrophoresis and then transferred from the gel to a two-dimensional support,
Recovery of Concentrated DNA Fragments from Pulsed Field Gels
DNA from low melting point agarose sections is first concentrated by electrophoresis into high percentage agarose gels and then separated by treatment with agar
Isolation of DNA from mammalian cells by the winding method
This method is a modification of the Bowtell method (1987), which allows for the simultaneous preparation of DNA from many different cell or tissue samples.The
Rapid isolation of yeast DNA
Yeast DNA prepared according to this protocol can be used as a template for PCR reactions. The shuttle plasmid, which replicates in both E. coli and Saccharomyc
Southern blotting (capillary transfer of DNA to a membrane)
Genomic DNA samples are prepared by digestion with one or more restriction endonucleases, and the digested fragments are separated by electrophoresis on a stand
Isolation of DNA from mammalian cells grown in 96-well microculture plates
This protocol, modified from the method of Ramirez-solis et al. 1992, 1993, is a simple and efficient method for extracting genomic DNA from eukaryotic cells gr
Preparation of genomic DNA from mouse tails or other small samples
This simple protocol is widely used in hundreds of laboratories for genotyping transgenic or knockout mice, and for extracting DNA from small amounts of culture
Recovery of DNA from low melting point agarose gels (organic solvent extraction)
Hydroxyethyl-modified agarose reduces the number of hydrogen bonds between chains and can melt and solidify at lower temperatures than standard agarose. The ext
Purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography
DNA fragments recovered from agarose gels, such as PCR products or digested DNA fragments, are resistant to further digestion. The reason for this resistance is
Growth of Saccharomyces cerevisiae and its DNA preparation
DNA prepared by this method is suitable for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that
Preparation of small quantities of yeast DNA
Yeast DNA can be prepared by digesting the cell wall and lysing the resulting protoplasts with SDS, and several milligrams of yeast DNA can be reproducibly prep
Isolation of genomic DNA fragment ends in high-capacity vectors (small vector PCR)
The genes of many eukaryotes contain far more DNA than a single recombinant can hold. This is especially true for most chromosomes. Therefore, it is necessary t
Detection of DNA in agarose gels
Nucleic acids in agarose gels can be detected by staining under UV light at a wavelength of 300 nm. Two methods for staining nucleic acids in agarose gels are i
Oligonucleotide 5' terminal phosphorylation assay
The following describes a reaction for labeling a 10 pmol high specific activity oligonucleotide. Labeling of different amounts of oligonucleotides can be accom
Purification of radiolabeled oligonucleotides by CPB precipitation assay
This protocol describes the separation of radiolabeled oligonucleotides from unadulterated radiolabeled material by quantitative differential precipitation with
Ligation experiments of λ phage arms to exogenous genomic DNA fragments
When a λ phage arm is attached to an exogenous genomic DNA fragment, two parameters must be taken into account: the molar ratio of the phage arm to the potenti
M13 Phage spread plates
M13 Phage plaques are formed when a single virus infects a single bacterium. The daughter virus particles infect neighboring bacteria and then produce the next
M13 Phage liquid culture
M13 Phage progenitors are usually grown in liquid culture, where infected cells do not lyse but grow slowly to form a dilute suspension. Inoculation is almost a
M13 Preparation of phage double-stranded (replicative) DNA
Bacteria infected with M13 phage contain viral double-stranded RF DNA, and single-stranded daughter DNA is contained in crude viral particles in the culture med
M13 Preparation of phage single-stranded DNA
M13 phage single-stranded DNA was prepared from viral particles secreted by infected cells into the surrounding culture medium. filoviruses were first concentra
Large-scale preparation of single- and double-stranded M13 phage DNAs
This protocol is mainly used to prepare large quantities of double-stranded DNA from M13 phage, which is often used as a cloning vector in the laboratory, as we
Determination of β-galactosidase in cellular extracts of experimental
This protocol describes the determination of expressed β-galactosidase activity after transfection of mammalian cells with the reported molecular vector (see F
M13 Cloning of phage vectors
Although there is theoretically no limit to the number of fragments of exogenous DNA that can be carried by a recombinant M13 phage, in practice there is a limi
Recombinant M13 phage cloning analysis
In this protocol, when the exogenous DNA is larger than 200~300 nucleotides, it can be identified by gel electrophoresis analysis of the single-stranded DNA rel
Preparation of single-stranded DNA using phage vectors
Phage plasmids skillfully combine the characteristics of plasmids and filamentous phages. In addition to the basic features, these plasmids are usually high cop
Phage DNA transfer experiments from phage spots to filter membranes
A method for identifying and isolating specific recombinants from λ phage libraries was established early in the history of molecular cloning by Bemon and Davi
Detection of DNA staining in polyacrylamide gels
Unlike agarose gels, ethidium bromide cannot be added to polyacrylamide gels as this dye interferes with the polymerization of acrylamide. However, ethidium bro
Long-range PCR
Under standard PCR reaction conditions, PCR amplification of DNA fragments is generally 1-2 kb in length, which is sufficient for many routine DNA molecular man
The cloned PCR product is ligated into a T vector.
DNA fragments amplified by Taq DNA polymerase PCR with a 3' prominent A base are efficiently cloned into T vectors that have an unpaired 3' T base complementary
Flat-end cloning of PCR products
After the target gene is amplified by PCR and the sample is purified and recovered as necessary, the next step is to use flat ends for ligation and cloning, whi
Construction of genomic DNA libraries using mucoid vectors
The steps for constructing genomic DNA libraries in λ phage are essentially the same as in mucosal vectors. In both systems, eukaryotic DNA is ligated to vecto
Phage DNA hybridization experiments on filter membranes
Filter membranes carrying immobilized DNA derived from phage spots can be screened by in situ hybridization with 32P-labeled probes. The technique is robust, hi
Simultaneous Preparation of DNA, RNA and Proteins from Cells and Tissues in a One-Step Process
Like the rapid RNA extraction method reported by Chomczynski and Sacchi in 1987, this method consists of lysing cells with a single-phase solution containing gu
Isolation of high molecular mass DNA from mammalian cells using formamide
This protocol is a modified version of the method described by Kupiec et al. (1987), which consists of digestion of cells and tissues with proteinase K, separat
Rapid analysis of λ phage isolates (purification of λ DNA from liquid cultures) Experiments
As described in this scheme, λ phage DNA can be easily purified from liquid cultures. In contrast, the previous scheme describes a method for purifying λ phag
Isolation of RNA according to size: agarose gel electrophoresis of glyoxalized RNA
This method combines glyoxal denaturation and agarose gel electrophoresis (modified from that of McMaster and Carmichael (1977), Thomas (1983)). automated glyox
P1 Application of phages and their cloning systems
The P1 phage was discovered in the same year as the λ phage (Bertani 1951). Both phages are mild phages in their natural hosts, and their research history in t
Recovery of DNA fragments from polyacrylamide gels (crush and soak method)
The standard method for recovering DNA from polyacrylamide gels is the "crush and soak" technique first introduced by Maxam and Gilbert (1977). The eluted DNA i
Isolation of high molecular mass DNA from mammalian cells using proteinase K and phenol
This procedure is adapted from a method first described by Daryl Stafford and colleagues (Blin and Stanfford 1976). This method should be used when large quanti
quantitative PCR
Quantitative PCR can be used for: (1) quantification of the amount of PCR starting template by analyzing the PCR end-product or monitoring the PCR process using
DNA recombination in vitro
DNA recombination, also known as gene cloning, is the most basic technique in gene manipulation and the core technology of molecular biology.DNA recombination r
DNA electrophoresis (agarose gel)
DNA electrophoresis can be used to (1) separate DNA fragments of different sizes, (2) identify target DNA fragments, and (3) purify and recover DNA fragments.
Differential display PCR
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Synthesis of non-fixed-length single-stranded DNA probes from M13 phage templates
During synthesis, low concentrations of radiolabeled dNTP limit the length of probes to 200-300 nucleotides. However, newly synthesized DNA can be made 100-1000
DNA barcode collection-analysis protocol experiment
The rise of biological barcoding demonstrates the active search for a system to identify eukaryotes based on the analysis of sequence diversity in short, standa
In vitro transcriptional synthesis of single-stranded RNA probes
Not only is it easier to prepare specific single-stranded RNA probes than DNA probes, but they also generally produce stronger signals in hybridization reaction
Radiolabeling of minus cDNA probes by random oligonucleotide extension method
The synthesis of cDNA in this protocol was carried out in four saturating concentrations of dNTP and one trace of radiolabeled dNTP. After deduction of hybridiz
Single-cell mRNA differential display assay
Several methods exist to show changes in the expression levels of unknown genes in tissue samples in response to physiological stimuli. However, the morphologic
Labeling the 3' end of double-stranded DNA with T4 phage DNA polymerase
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Comparative Molecular Physiological Genomics - Heterozygous hybridization experiments with cDNA arrays
In recent years, DNA microarrays have been recognized as a standard method for molecular biology research. Especially in biomedical research, microarrays of com
Phosphorylation of DNA molecules containing a 5' prominent hydroxyl terminus
This protocol removes the 5' phosphate of a nucleic acid by phosphatase, and then re-adds the phosphate to the nucleic acid in a radiolabeled form catalyzed by
Toxicological effects of environmental toxins detected by cDNA macroarrays and gene expression profiles
This chapter describes how to prepare and use cDNA macroarrays to detect the effects of environmental toxins on gene expression, and describes the materials and
Anchoring enzyme digestion of cDNA
If the previous step in the series of analytical techniques for gene expression, SAGE cDNA, was stored at -20°C, then this experiment was thawed slowly on ice.
Phosphorylation of 5' prominence DNA molecules by exchange reaction
The T4 phage polynucleotide kinase-catalyzed exchange reaction (Van de Sande et al. 1973; Berkner and Folk 1977) is a rapid method for labeling 5' end DNA. Unli
Experimental isolation of ozone-responsive genes from Arabidopsis thaliana by the cDNA macroarray method
In addition to cDNA microarrays, cDNA macroarrays based on nylon membrane supports are another widely used method for collecting large-scale gene expression dat
Agarose gel electrophoresis experiment
Agarose gel electrophoresis can be applied to (1) DNA cutting and recovery, (2) DNA isolation, (3) corroboration of whether DNA is recombinant or not, whether p
Synthesis of cDNA probes from mRNA using random oligonucleotide primers
In this protocol, poly(A)+ RNA is used as a template to synthesize cDNA probes by random priming reaction, and these probes can be used for differential screeni
In-situ notch panning technique
The in situ nick translation technique is also known as in situ nick translation. It was first used to study DNA replication in vitro, and has since been extend
Phosphorylation of dephosphorylated flat-end or 5' concave-end DNA molecules
In the forward reaction of T4 polynucleotide kinase, DNA substrates with flat ends, 5' concave ends or intramolecular cuts are less efficient than molecular lab
Microbial Genome DNA Extraction
The yeast is first lysed using a solution containing leaving salts to ensure complete denaturation of the macromolecules. Afterwards, ethanol is added and the D
Automated Laser Fluorescence DNA Sequencing
At present, the application of automated laser fluorescence DNA sequencing technology (also known as first generation sequencing technology) has been very commo
Micronucleus assay for detecting DNA damage at the chromosome level
The study of DNA damage at the chromosomal level is an important part of genotoxicology because chromosomal mutations are an important part of the cancer proces
Determination of DNA concentration and purity
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Pyrophosphate sequencing
Pyrosequencing is a luminescence-based sequencing technology for the determination of pyrophosphate (PPi). The three key points of pyrosequencing are: (1) dATPa
Mutagenesis of genomes using chemical mutagens
Currently, there are two main strategies for studying gene function using model mice: the gene-driven approach and the phenotype-driven approach. Gene knockout
Synthesis of fixed-length single-stranded DNA probes from M13 phage templates
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Quantitative analysis of trace DNA by PCR method
PCR is used to quantify the amount of a particular DNA sequence from 1 to 20,000 molecules per sample. In addition, it can assist in assessing the presence of c
Synthesis of radiolabeled minus cDNA probes using oligo (dT) as primer
This protocol describes the preparation of a deduction cDNA probe by hybridization with an mRNA driver followed by purification of single-stranded radiolabeled
Southern blotting technology
Blot hybridization is the process of binding a nucleic acid fragment to be tested to a certain solid phase support and then hybridizing it to a labeled probe pr
Establishment of an insect baculovirus expression system
The baculoviruses are double-stranded DNA viruses that infect mainly insects, but also in vitro insect cell lines, and are not toxic to humans or animals. Alfal
Poxvirus DNA extraction experiment
If extracted poxvirus DNA is used for transfection, it should be isolated after proteinase K digestion and phenol extraction. See this experiment for specific m
Isolation of RNA based on size
RNA samples may be denatured by treatment with formamide and by electrophoretic separation on gels containing formaldehyde, a method modified from Lehrachetal.
Dephosphorylation of DNA fragments by alkaline phosphatase
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Related topics
Techniques often explored alongside immunological experiments.