DNA experiment

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

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

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

Formaldehyde-agarose gel electrophoresis (formaldehyde-agarose gel electrophoresis) Formaldehyde is a commonly used denaturant for RNA. It is used for (1) RNA i

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

Determination of DNA content can be used to (1) perform gene fragment ligation experiments. (2) Perform plasmid concentration determination prior to transformat

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

SNP (Single Nucleotide Polymorphism), or Single Nucleotide Polymorphism, can be used to detect nucleic acid sequence polymorphisms (Polymorphism) due to changes

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

Subcloning of the target gene can be applied to (1) further analysis of the target gene and (2) gene recombination.

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

Denaturing gradient gel electrophoresis (DGGE) is a gel system that enables the separation of DNA fragments according to their melting properties. (1) By settin

DNA sequence analysis techniques can be used to (1) analyze the genetic diversity of species, (2) identify new species, and (3) for comparative genomics.

This experiment is mainly used to obtain eukaryotic cells containing the target exogenous gene.

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

Bisulfite-modified sequencing can be used primarily to detect methylation.

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

Recombinant plasmid ligation, transformation and screening are mainly used to obtain recombinants containing the target gene linkage.

Pulsed-field gel electrophoresis, which alternates periodically between two differently oriented electric fields, allows the separation of DNA molecules up to 5

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

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

Expression experiments of multidrug resistance genes are mainly used for tumor therapy research.

Region-specific mutagenesis experiments for (1) gene-induced modification (2) specific expression of genes (3) clinical anti-tumor and other targets.

Gene synthesis can be applied to (1) metabolic pathway synthesis, (2) gene network construction, and (3) vaccine design.

The mutated oligonucleotide is used to direct the synthesis of the template, thereby altering the DNA sequence, with a mutation efficiency of 50-80%.

An important feature of this method is the direct cloning of single-stranded, abbreviated oligonucleotides into homologous double-stranded molecules after their

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

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

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

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

Most commonly used plasmids contain multiple cloning sites that can be recognized by different endonucleases. Because of the large number of cloning sites avail

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

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Source of content: compact guide to molecular biology 5th ed.

Source of content: Guangdong Pharmaceutical University Laboratory Instruction Manual.

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

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

In the modification of DNA fragments, the dephosphorylation reaction is an important element, which is catalyzed by alkaline phosphatase, an enzyme that removes

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

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

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

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

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

When Sanger and colleagues established the first DNA strand termination extension method, there was only one suitable DNA polymerase available, the Klenow fragm

The importance of chemical sequencing has always been demonstrated by obtaining sequences of oligonucleotides, functional analysis of transcriptional regulatory

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

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

In sequencing, 4 series of DNA fragments are analyzed by electrophoresis on a thin polyacrylamide gel under denaturing conditions. This protocol describes the p

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

This experiment describes the procedure regarding radioactive autoradiography and the reading of DNA sequences from sequencing gels. This experiment comes from

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

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

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

The classical Kunfcel oligonucleotide-directed mutagenesis method utilizes the selective action of uracil DNA glycosylase in E. coli to specifically screen out

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

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

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,

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

Electroshock transformation experiments in E. coli can be used for: easier and faster transformation.

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

This protocol mainly describes the screening of recombinant phage M13 clones, whereas one option is to screen bacterial clones containing phage particles. Final

This protocol uses the nuclease BAL31 (purified from the marine bacterium Alteromonas espejiana BAL31) to produce unidirectional or bidirectional deletions in c

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

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

There are three methods for detecting bound antibodies: radiochemical screening, colorimetric screening, and chemiluminescent screening, the advantages and disa

This protocol describes a method for removing antibodies reactive with bacterial-encoded proteins from polyclonal antisera by co-warming the antiserum with bact

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

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

This section describes a method for removing components of crude immunoglobulin that react with bacterial-encoded proteins using a matrix that binds bacterial p

This protocol is suitable for the preparation of phage progenitors, the preparation of phage particles (used to obtain DNA for subcloning), and the preparation

This experiment describes the process of expressing exogenous proteins in Escherichia coli using the alkaline phosphatase promoter (phoA) and signaling sequence

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

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

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

Fusion proteins encoded by λ phage recombinants can be prepared by lysogenizing colonies of E. coli strain Y1089. However, preparation of lysogenic bacteria fr

This protocol will discuss the factors affecting plasmid expression efficiency in the Troubleshooting and Optimization of Inducible Promoter Expression Proteins

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

This method is suitable for rapid screening of immunodetectable fusion proteins against λgtll recombinant phage. However, under other conditions (e.g., optimiz

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

This experiment was mainly used to obtain soluble active proteins.

The following method is a modification of one studied by Mark Evans (Alexion Pharmaceuticals, New Haven, Connecticut). This experiment was derived from the next

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

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

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.

Affinity chromatography methods can virtually purify maltose-binding fusion proteins into single components. Maltose-binding protein self (MBP) is a periplasmic

The exogenous protein expressed in the pGEX vector is fused to glutathione S transferase and can therefore be purified by glutathione-agarose affinity chromatog

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

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

DNAase I hypersensitive site mapping is frequently used to localize regulatory regions of eukaryotic genes. For reasons that are not yet understood, genes carry

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

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

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

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

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

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

Thick bacterial colonies transformed by mucilage can be screened by hybridization, a method derived from the large-scale screening of plasmid-transformed coloni

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

Nuclear contig analysis is used to determine whether a gene is transcriptionally regulated in a given clone. Theoretically, nuclear contig analysis should be pe

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

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

The following protocol is modified from the protocol that accompanies the Ecdysone-Induced Mammalian Expression System (Invitrogen); a similar system is sold by

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

GST fusion proteins, as recombinant proteins expressed in bacteria, have been used in thousands of research projects since their introduction (Smith and johnson

This experiment describes experimental methods regarding two-hybrid and other two-component systems. This experiment comes from the next volume of the Laborator

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

Applying this amplification method, the library is not prone to distortion, due to the fact that mixed colonies containing different recombinant mucilages do no

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

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

BIAcore surface plasmon resonance is widely used for (1) studying the interaction process between various biomolecules (e.g., peptides, proteins, oligonucleotid

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

The method detailed in this protocol for the purification of crude synthesized oligonucleotides using denaturing polyacrylamide gel electrophoresis is a modific

When radiolabeled oligonucleotides are used in enzymatic reactions such as primer extension reactions, it is necessary to completely remove the unadulterated ra

If the radiolabeled oligonucleotide is to be used only as a hybridization probe, it is generally not necessary to completely remove the unadulterated radiolabel

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

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

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

Jabobs et al. (1988) described the method and principles of hybridization in quaternary ammonium-containing buffers. The following is a simple variation of this

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

This scheme describes a method to separate radiolabeled oligonucleotides from undoped radiolabel using the property of antiphase affinity of oligonucleotides fo

The unlinking temperature for hybridization of oligonucleotides to target sequences can be calculated using the method described in the overview of this chapter

Recovery of DNA from agarose gels can be applied to (1) mammalian cell transformation and (2) radiolabeling.

Oligonucleotides are usually labeled using a phosphorylation reaction catalyzed by the T4 phage polynucleotide kinase. However, there is sometimes a need to lab

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

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

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

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

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

In clamped homogeneous electric field (CHEF) gel electrophoresis, the electric field is generated by multiple electrodes. These electrodes are arranged in a qua

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

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

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

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

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

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

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

Ribonuclease protection analysis is used to measure the abundance of specific mRNAs and to map their topological heterogeneity. This method involves hybridizati

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

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

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

RT-PCR can be used to: (1) construct large-capacity cDNA libraries; (2) identify mutations and polymorphisms in transcribed sequences; (3) measure the intensity

This protocol (provided by Andereson of the University of Texas Southwestern Medical Center) describes the introduction of restriction endonuclease sites at bot

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

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

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Using oligonucleotides as primers, DNA polymerase can initiate DNA synthesis along a single-stranded template (Gotilian 1969). If the sequences of the oligonucl

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

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

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

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

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

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

SAGE (Velculescuetal.1995) is a highly sensitive method based on PCR technology to study gene expression and to obtain qualitative and quantitative information

Electron microscopy in situ hybridization experiments can be used to (1) study the subcellular localization of specific nucleotide sequences; (2) many researche

Materials Many organisms are excellent models for disease studies or for exploring the molecular adaptive mechanisms that enable cells and organisms to cope wit

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

Modern Neuroscience Research Techniques Author(s): U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun

M-FISH has been successfully used to identify marker or derived chromosomes in congenital diseases. Recently, this method has been increasingly used to identify

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

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

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

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

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-

Fluorescence in situ hybridization (FISH) technology allows the detection of specific nucleic acid sequences in chromosomes, cells and tissues. The unambiguous

BAC (Bacterial Artificial Chromosome) libraries can be used for (1) whole genome sequencing; (2) construction of physical maps, chromosome step-by-step; (3) gen

The interspecies color banding technique is a simple and rapid method for detecting chromosomal abnormalities that cannot be identified by G-banding. This techn

Chromosomal aberrations are usually detected in most hematological and various solid tumors and, clinicopathologically, are frequently associated with the deter

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

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

There are two commonly used nucleic acid electrophoresis methods. ① Horizontal electrophoresis: agarose gel, bands are judged under UV light. This method is ec

Gene amplification is frequently detected in human tumor cells and plays an important role in tumorigenesis. Genome-wide scans of tumor cells using comparative

Fluorescence in situ hybridization (FISH) techniques enable rapid detection of chromosomal abnormalities in a variety of tissues, including fresh and archival s

Differential cloning is an effective technique for isolating and characterizing mRNAs that are differentially expressed in two cell populations. The cell types

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

Source : Molecular Cytogenetics Techniques and Applications

Source : Molecular Cytogenetics Techniques and Applications

Source : Molecular Cytogenetics Techniques and Applications

Source: Molecular Cytogenetic Technology and Applications

Rapid amplification of cDNA ends

Luciferase plasmids were constructed and then transfected into cells to detect alterations in reporter gene expression by comparing overexpression or interferen

Methylation testing of DNA

Animal liver DNA extraction can be (1) used for forensic identification, (2) for diagnostic and phylogenetic studies, and (3) for further polymerase chain react

Purification of plasmid DNA from bacteria

Non-banded chromosomes are difficult to identify except for some chromosomes based on their morphology, whereas the banding technique allows the appearance of d

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

M13 phage is a filamentous phage that does not lyse bacterial cells after infection, but only utilizes intracellular material to complete its own proliferation,

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 can be used for: (1) after extracting the total RNA of the sample, the quality of RNA is generally judged accor

Nucleic acid probe molecular hybridization refers to the two nucleic acid single strands with certain homology can be formed under certain conditions according

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

Bacterial genomic DNA extraction can be applied to: (1) obtain bacterial genomic DNA; (2) as PCR template; (3) for sequencing, genetic information analysis, etc

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

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

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

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

The experiment of precipitating DNA can be applied to isolate DNA.

Gene transfection technology can be applied to: genome function research (gene expression regulation, gene function, signal transduction and drug screening rese

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

Radioisotope-labeled DNA sequencing analysis can be applied to analyze gene structure-function relationships.

The freeze-thaw method of recovering DNA fragments can be used to (1) obtain purified DNA fragments and (2) recover PCR products.

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

Chemical determination of DNA content can be used for (1) estimation of plasmid concentration prior to transformation and transfection, and (2) assay of acquire

The polymerase chain reaction constructs recombinant DNA for a wide range of uses (1) for sequencing primers (2) targeted mutagenesis (3) nucleic acid hybridiza

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

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,

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

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

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

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

To remove ethidium bromide from CsCl-gradient purified DNA, repeated extraction with organic solvents is commonly used. Subsequently, CsCl can be removed by dia

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

Ethidium bromide in DNA purified by cesium chloride gradient centrifugation can be removed by ion-exchange chromatography, followed by ethanol precipitation to

Standard double deoxy DNA sequencing can be easily and efficiently detected using non-isobaric labeled chemiluminescence. In the sequencing reaction, biotin-lab

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

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

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

Restriction endonucleases are a class of DNA hydrolases that recognize specific nucleotide sequences in double-stranded DNA molecules, and the discovery and app

DNA molecule is the basic material of molecular biology research, according to different experimental purposes can be taken to different extraction methods to o

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

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

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

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.

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

In typical immunoscreening experiments, libraries of λ phage expression vector constructs should be spread on plates of E. coli strains that do not conta

Phages originate from the infection of a single bacterium by a single phage particle. The daughter virus particles synthesized after the first infection adsorb

This protocol describes how to hybridize radiolabeled probes to DNA of transforming bacteria immobilized on filter membranes, and methods for recovering clones

Using this scheme, well-sheared, high-quality salmon sperm stretcher DNA can be obtained, which can be used in transformation experiments and other experiments

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

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

Genetically homologous λ phage protospecies are obtained by "picking" a completely separate phage spot and preserving agar/agarose containing the lysate

This experiment was used to detect sequence-specific DNA-binding proteins in crude extracts.

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

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

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

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

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

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

Replacement vectors (e.g., λ2001, λDASH, EMBL series, Charon 34, Charon 35, and Charon 40) contain a series of restriction sites that are aligned

Mass preparation of λ phage can be obtained by either low-mass infection of bacterial cultures or high-mass infection. Immediately after low magnificatio

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

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,

Synthetic double-stranded oligonucleotides can be used to screen cDNA expression libraries constructed in λ phage to identify clones corresponding to spe

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

Phage plasmids skillfully combine the characteristics of plasmids and filamentous phages. In addition to the basic features, these plasmids are usually high cop

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

This protocol describes the determination of expressed β-galactosidase activity after transfection of mammalian cells with the reported molecular vector (see F

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

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

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

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

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

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

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 phage single-stranded DNA was prepared from viral particles secreted by infected cells into the surrounding culture medium. filoviruses were first concentra

This protocol describes the separation of radiolabeled oligonucleotides from unadulterated radiolabeled material by quantitative differential precipitation with

The following describes a reaction for labeling a 10 pmol high specific activity oligonucleotide. Labeling of different amounts of oligonucleotides can be accom

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

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

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

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

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

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

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

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

Genomic DNA samples are prepared by digestion with one or more restriction endonucleases, and the digested fragments are separated by electrophoresis on a stand

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

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

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

DNA prepared by this method is suitable for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that

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

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

Hydroxyethyl-modified agarose reduces the number of hydrogen bonds between chains and can melt and solidify at lower temperatures than standard agarose. The ext

Unlike agarose gels, ethidium bromide cannot be added to polyacrylamide gels as this dye interferes with the polymerization of acrylamide. However, ethidium bro

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

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

This method combines glyoxal denaturation and agarose gel electrophoresis (modified from that of McMaster and Carmichael (1977), Thomas (1983)). automated glyox

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

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

DNA from low melting point agarose sections is first concentrated by electrophoresis into high percentage agarose gels and then separated by treatment with agar

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,

Three different nucleases, S1 nuclease, RNAase, and exonuclease VII, are used to quantify RNA, to identify intron positions, and to characterize the positions o

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

The signal intensity obtained by Southern hybridization depends on several factors, including the ratio of immobilized DNA complementary to the probe, the size

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

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

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

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

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

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

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

Radioactive DNA bands separated by acrylamide gel electrophoresis can be detected by radioautography. Polyacrylamide analytical gels containing radioactive DNA

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

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.

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

In addition to cDNA microarrays, cDNA macroarrays based on nylon membrane supports are another widely used method for collecting large-scale gene expression dat

In recent years, DNA microarrays have been recognized as a standard method for molecular biology research. Especially in biomedical research, microarrays of com

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

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

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

Several methods exist to show changes in the expression levels of unknown genes in tissue samples in response to physiological stimuli. However, the morphologic

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

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

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

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

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

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

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

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

This protocol describes the preparation of a deduction cDNA probe by hybridization with an mRNA driver followed by purification of single-stranded radiolabeled

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

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

The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

At present, the application of automated laser fluorescence DNA sequencing technology (also known as first generation sequencing technology) has been very commo

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

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

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

Currently, there are two main strategies for studying gene function using model mice: the gene-driven approach and the phenotype-driven approach. Gene knockout

Pyrosequencing is a luminescence-based sequencing technology for the determination of pyrophosphate (PPi). The three key points of pyrosequencing are: (1) dATPa

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

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 can be used to (1) separate DNA fragments of different sizes, (2) identify target DNA fragments, and (3) purify and recover DNA fragments.

RNA samples may be denatured by treatment with formamide and by electrophoretic separation on gels containing formaldehyde, a method modified from Lehrachetal.

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

None

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

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