Why Johnny can't clone: Common pitfalls and not so common solutions.
The demand for cloned genes has increased incessantly over the past 32 years, but some who need recombinant plasmids struggle to produce them. While the pitfalls of traditional ligation-dependent cloning are non-trivial, most can be avoided with sufficient effort and attention to detail. Here, the chemical properties of enzymes and reagents used to clone genes into plasmids are reviewed to draw attention to the most pertinent details. In particular, the virtues of agarose gel electrophoresis monitoring, the nature of the interactions between DNA and silica, and challenges associated with thermostable DNA polymerases, restriction endonucleases, and T4 DNA ligase are explored. Common pitfalls associated with Escherichia coli transformation and DNA modifying enzymes are also described. A thorough understanding of established methods is essential for troubleshooting, implementing alternative approaches, and inventing new techniques in response to changes in technology and demand.
Department of Biochemistry, Emory University School of Medicine, Rollins Research Center, Atlanta, GA.
Ichiro
I
Matsumura
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation / Q000379:methods; D004798:Enzymes / Q000378:metabolism; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods
D004798:Enzymes; D004247:DNA; D004262:DNA Restriction Enzymes; D011088:DNA Ligases
DNA purification; PCR; T4 DNA ligase; electrophoresis; ligation dependent cloning; molecular cloning; restriction endonuclease