The basic procedure performed by any molecular biologist is, naturally, cloning. You may wish to clone a DNA fragment into a plasmid for many purposes, such as protein expression and purification, gene knock out / silencing / overexpression, reporter gene fusion to a gene of interest, and many more. Whether you plan on doing one thing or another with the plasmid you cloned, when it comes to the cloning procedure you will need to perform the same steps, which are the ABC’s of molecular biology. However simple it may seem, when you don’t have enough experience in cloning plasmids, you can find yourself struggling endlessly, going over the same protocol over and over again for weeks and even months, gaining no success or insights as to what went wrong. I’ve seen so many frustrated graduate students and postdocs walking around the corridors of research buildings, knocking on neighboring lab doors looking desperately for help and advice. I certainly do not claim to be a cloning guru, on the contrary. I’ve had my fair share of nightmare-haunted nights, full of dreams featuring linear vectors unwilling to circularize, and images of me in front of the PhD committee having nothing to discuss. It took me quite a while (and many frustrations and disappointments) to realize that before jumping into the icy cold water of molecular biology protocols, I need to thoroughly read, learn and consult experts. It’s as simple as that: the more you know about the cloning protocols, understand the rationale behind each step, and the function of each component used, the faster and easier you’ll cross the bridge from preparing the molecular tools for your research to the actual science making.
We therefore prepared here for you, the molecular biologist, a step-by-step protocol for cloning plasmids. For each step you’ll find some useful suggestions and recommendations. The procedures discussed in this protocol include:
- Polymerase chain reaction
- Bacterial transformation
- Plasmid DNA isolation
- And more
You are invited to download and use our whitepaper as a basic guide to your cloning.
Good luck with your cloning!