Restriction Enzyme Cloning Manual

Molecular Cloning is a set of techniques that are used by molecular biologists to insert a gene-of-interest into a vector capable of replication within the target cell. Once the gene is being expressed in the cells, the system can then be used for a number of different research applications and studies.

An early method that is still of widespread use is restriction enzyme cloning. The method is fairly inexpensive and quite reliable, although it does require some forethought as the researcher is engineering something new and specific to them, for their independent use. The Restriction Enzyme Cloning Manual, provided by Oxford Genetics, aims to assist you through the basic protocols to build and test your expression vector.

Cloning the Gene-of-Interest into a Plasmid Vector

• Selecting the Cloning System and Plasmid Vector
• Plasmid Restriction Digestion
• Fragment Restriction Digestion
• Gel Excission
• Clean-up of Gel Fragments
• Annealing DNA Oligos for Ligation
• Adding 5’ Phosphates to DNA
• DNA De-phosphorylation
• Preparing DNA for Bunt-ended Cloning
• Ligating the DNA to yield a plasmid containing the Gene-of-Interest
• Site-Directed Mutagenesis

Transforming E. coli with Engineered Plasmid

• Making Competent Cells
• Making Agar Plates
• Bacterial Transformation
• Picking Colonies
• Growing Bacteria in Liquid Culture
• Freezing Bacteria

Selecting Correctly Expressing Recombinants

• Blue White Screening
• DNA Minipreps
• Screening by Restriction Digestion
• Screening by PCR
• Confirm cut plasmid sizes by agarose gel electrophoresis
  
• DNA Maxipreps
• DNA Precipitation
• RNase Treatment
• Clean-up of DNA

Buffer Recipes

Glossary

Vector choices don’t have to be complicated. Using SnapFast™ Technology built into expression vectors by Oxford Genetics, you get a new standardized tool for your molecular biology toolkit. Exchange DNA components with simple restriction digests to build the plasmid you need for your research. We have everything you need for your molecular cloning work.

Looking for more vector options to move your experiments forward faster? Consider a custom cloning vector designed and built by Oxford Genetics™. Find out more at Oxford Genetics - our partner for cloning and expression vectors for molecular biology and synthetic biology applications.

About Oxford Genetics

Oxford Genetics specialises in the production of modular DNA plasmids for biological research. Their main technology (SnapFast™) is designed to make genetic engineering cheaper and more efficient by providing scientists with a versatile cloning platform and access to a library of matched DNA inserts. Our concept is simple: One plasmid, a thousand possibilities.