Difference between revisions of "Resources/Troubleshooting"

Introduction

Cloning is difficult. No one who has ever picked up a pipette in the lab would debate this fact. However, cloning shouldn't be impossible! This page exists to help students and teams who are struggling with cloning and other molecular techniques. Treat this page as another resource in your troubleshooting endeavors rather than the be-all, end-all resource for iGEM cloning problems. This is an experimental project for the 2015 iGEM season and I hope this will be a helpful resource for every iGEM team!

As the Science and Technology Fellow at iGEM Headquarters, I want to try to help you out with cloning problems to the best of my abilities. To this end, I will be offering advice and possible solutions to your problems. As I start collecting questions and generating answers, I plan to create a Troubleshooting Guide that will serve as a resource for both current and future teams to use throughout their iGEM experience.

My background is primarily in bacterial cloning with a focus on working in E. coli. I completed my doctorate in cell and molecular biology from the University of Rhode Island in 2011 and was a postdoctoral researcher in synthetic biology with Dr. Douglas Densmore at Boston University from 2011-2013. From there, I continued to work in the lab as the Executive Director of the Boston University Center of Synthetic Biology and I joined the iGEM Headquarters team in April 2015. In summary, I have over 14 years of experience with molecular techniques and cloning in E. coli, and over 4 years of experience with synthetic biology in particular.

General Tips and Tricks

This section is meant to provide some general advice for cloning problems. I recommend you read through this material if you're having problems while you're waiting for a response to your specific question. Each link below is geared towards a specific area where cloning problems occur.

General Transformation Issues
For many teams, cloning problems center around transformation issues. Here I describe some general tips that can be applied to any type of assembly method when using E. coli cells as the transformation host.

• E. coli Transformations

General BioBricks Cloning Problems
Since we provide teams with nearly 2,000 BioBricks parts, I will discuss common problems students can experience when using BioBricks assembly. However, these tips and tricks can also be used for other assembly strategies that utilize restriction enzymes and DNA ligation.

• Restriction Digest and Ligation

Note:If you have other tips you'd like me to include in these pages, please send them using the question form below! Likewise, if you tried out one of the tips and it worked (or didn't work), please let me know through the form below or by emailing me at traci AT igem DOT org. Your feedback will help us track how useful this section is so we can improve it throughout the competition.

iGEM Protocols

Below are the links to the various protocols that iGEM Headquarters has provided over the years. These protocols can also be found through the Parts Registry page under the "Help" and "Protocols" links in the black toolbar at the top of the page.

These are another useful resource that teams should read through when they're experiencing cloning problems.

BioBrick Cloning

• 3A Assembly
• Prefix and Suffix
• PCR standardization
• Linearized plasmid backbone
• Restriction digest

Cloning protocols that can be applied to BioBricks and non-BioBricks assemblies

• Making competent cells
• Antibiotic stocks
• Miniprep
• Ligation
• Transformation
• Transformation efficiency kit