Molecular Cloning Manual

[Ene Vinson]

Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems.

For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories.

Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled.

The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes.

Building on thirty years of trust, reliability, and authority, the fourth edition of Molecular Cloning is the new gold standard—the one indispensable molecular biology laboratory manual and reference source.

This Bootcamp is intended to provide undergraduate students with a technical and conceptual knowledge of basic techniques in molecular biology. The experiments and resources in this Bootcamp are designed to introduce students to many of the essential skills and techniques that are used every day in real research labs. The Bootcamp manual includes fundamental protocols for molecular cloning and protein purification, as well as additional resources for students and Bootcamp instructors.

Upon completion of this Bootcamp, students should have a solid foundation in the core techniques of molecular biology and essential skills of general scientific research. Students should feel more comfortable working in a research lab and more confident in their knowledge of these techniques and their fundamental principles.

The goal of this Bootcamp is to clone green fluorescent protein (GFP) fused to a 6X histidine (His6) tag into a plasmid vector for expression in the bacteria Escherichia coli. We will then isolate GFP protein from an E. coli extract using the His6 tag and use different methods to analyze the isolated GFP protein.

Many of the experiments in this course are used in sequence in a research lab to create or modify a desired DNA construct for use in experiments. Because of the time constraints of this course, the protocols we will follow are not all in the same order that you might experience if you were doing a similar project in a lab.

We will use Gibson Assembly to insert GFP into a plasmid vector (pET28b) that can be used to express 6X His-tagged proteins in bacteria. The protocols for this section can be found on the Molecular Cloning Overview page.

The pET28b-GFP construct we made in Part 1 contains an IPTG-inducible GFP protein with a 6X His tag. The protocols for this section can be found on the Protein Expression, Purification, and Analysis Overview page.

Expressing 6X His-GFP in E. coli requires a properly constructed plasmid. In an actual research project, completion of Part 1 is necessary before starting any of the steps outlined here. For the purposes of this class, we have already constructed the pET28b-GFP plasmid and transformed it into a protein expression strain of E. coli called BL21 DE3.

From here, experiments will vary based on individual research interests. Purified proteins are often used in biochemical assays to determine their functions or crystallized to determine their structure. What we do with a purified protein depends on the particular research questions, techniques available, and the properties of the protein.

The lectures and protocols in this course touch on some of the technical aspects of the assays you might do. As you will see through reading scientific literature and discussing with your research mentors, the specific assays you will use will need to be tailored to the specific questions you want to ask.

Blog Series: Learning by the Book

Join the conversation on Twitter with the hashtag #lbtb18 or on this blog with a post of your own. Tweet or email us links to related discussions. Read more posts in this series, and check out the conference website.

In 1973, a group of scientists at UCSF and Stanford, led by Herbert Boyer and Stanley Cohen, succeeded in placing a copy of a frog gene (one that encoded ribosomal RNA) into a bacterial plasmid. Not only was the inserted gene on its plasmid vector taken up and replicated by E. coli, but also the foreign DNA was expressed into the corresponding product RNA. Their 1974 publication became the much-cited proof that genes from a higher organism could be cloned and expressed in a bacterium.

Few molecular biologists welcome publication of any of the many protocol books that promise to be the single source for their laboratory methods. For the most part, such laboratory methods fall far short of this goal. So why the excitement surrounding the long-awaited second edition of the classic guide, Molecular Cloning, which first appeared in 1982? The original version immediately filled the need for an anthology of laboratory procedures pertinent to the emerging field of recombinant DNA. With the 545-page spiral-bound paperback in hand, virtually any experimentalist could make a stab at cloning and have a reasonable expectation of success.[16]

Molecular biology concerns the molecular basis of biological activity between biomolecules in the various systems of a cell, including the interactions between DNA, RNA, and proteins and their biosynthesis, as well as the regulation of these interactions.

One of the most basic techniques of molecular biology to study protein function is molecular cloning. In this technique, DNA coding for a protein of interest is cloned using polymerase chain reaction (PCR), and/or restriction enzymes into a plasmid (expression vector). A vector has 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a selective marker usually antibiotic resistance. Located upstream of the multiple cloning site are the promoter regions and the transcription start site which regulate the expression of cloned gene. This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation via uptake of naked DNA, conjugation via cell-cell contact or by transduction via viral vector. Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. Several different transfection techniques are available, such as calcium phosphate transfection, electroporation, microinjection and liposome transfection. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection.

DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.

We will use gel electrophoresis to separate the DNA fragments obtained from the restriction digest (Figure 12.1). Before setting up the digest, we will pour agarose gel because it will take about half an hour for the gel to harden.

We added a non-toxic green fluorescent dye to the agarose before we poured the gel. The inclusion of this dye will allow us to visualize the separated DNA fragments by exposing the gel to UV light source in the UV light box (Figure 12.3).

This video explains the major methods that are combined, in tandem, to comprise the overall molecular cloning procedure. Critical aspects of molecular cloning are discussed, such as the need for molecular cloning strategy and how to keep track of transformed bacterial colonies. Verification steps, such as checking purified plasmid for the presence of insert with restrictions digests and sequencing are also mentioned.

Molecular cloning is a set of techniques used to insert recombinant DNA from a prokaryotic or eukaryotic source into a replicating vehicle such as plasmids or viral vectors. Cloning refers to making numerous copies of a DNA fragment of interest, such as a gene. In this video you will learn about the different steps of molecular cloning, how to set up the procedure, and different applications of this technique.

At least two important DNA molecules are required before cloning begins. First, and most importantly, you need the DNA fragment you are going to clone, otherwise known as the insert. It can come from a prokaryote, eukaryote, an extinct organism, or it can be created artificially in the laboratory. By using molecular cloning we can learn more about the function of a particular gene.

3a8082e126