Learn about cloning troubleshooting here!
- Read about General Tips and Tricks with a focus on Transformations, Restriction Digests, and Ligations
- Have a cloning problem? Ask a question!
- See the Answers to Your Questions (which will be updated throughout the iGEM season!)
- While we want teams to use this resource, also make sure that you talk to your local resources as well - ask your instructors and more experienced students around you!
Welcome to the Troubleshooting page!
My name is Traci, and I'm here to help you with your cloning problems.
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.
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.
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.
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.
Cloning protocols that can be applied to BioBricks and non-BioBricks assemblies
- Making competent cells
- Antibiotic stocks
- Transformation efficiency kit
Answers to Your Questions
As teams start to submit their questions, I'll be posting answers here.
I'll also make sure to Tweet whenever I update this page, so please follow me on Twitter: @Traci_Haddock
Ask Your Questions and Submit Comments Here!
First and foremost, there are no stupid questions, especially when it comes to cloning problems. Ask me literally anything about cloning! I'm also happy to answer questions about general molecular biology techniques (including plasmid minipreps, DNA gel electrophoresis, and so on).
If you have other tips and tricks you'd like me to include, please send those comments using this form. In particular, we'd love to expand this section to include tips and tricks for non-E. coli chassis!
Finally, please send me any feedback about how useful this page was for your team. I would love to have feedback after you tried any of these suggestions to see what worked for your team. This type of feedback will help us improve this page throughout the competition!
Your name, team, and email will be kept confidential, but I will repost your question when I answer it. I'm asking that everyone provide a first name and an email so I can follow-up with you if your question or comment is unclear. Emails will come from traci at igem dot org if I need to follow-up with you.
Thanks very much for participating!
Important: If you have trouble using the form below, please send me an email at traci AT igem DOT org and ask your question! We will be updating this form shortly to be a more universally accessible form so all teams can submit their questions and comments.