Troubleshooting

Page is under construction.
iGEM HQ is currently working on updating this information for the iGEM 2015 competition.

Learn about cloning troubleshooting here!

  • Read about General Tips and Tricks
  • 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.

You can contact me by email at traci AT igem DOT org, on Reddit Traci_at_iGEM, or on Twitter @Traci_Haddock

Introduction

Cloning is difficult. No one who has picked up a pipette in the lab would debate this fact. However, cloning shouldn't be impossible! This page exists to help 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

Transformation Problems

One of the most common problems that researchers have in the lab is transformation efficiency. Have you done transformations but have seen few to no colonies on your plates? You're not alone! Here are some tips to help you troubleshoot this problem:

  • How competent are your cells? If you don't know the answer to this question, you need to run a positive control and calculate your efficiency!
    1. Transform your cells with a known quantity of plasmid. iGEM provides each team with a Transformation Efficiency Kit for this exact purpose. You can also use your own plasmid if you know the concentration of the DNA.
      Important: You must use plasmid for calculating transformation efficiency. If you use a ligation product, your calculation will be incorrect due to the low amount of circularized DNA in a ligation reaction.

    2. Calculate your transformation efficiency with the following equation:
      (# colonies on plate/ng of DNA plated) X 1000 ng/µg = colony forming units/µg of DNA

    3. The measurement "ng of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:
      volume of plasmid used in µL x concentration of DNA x (volume plated / total reaction volume)

    4. Example: You transformed 1µL of 0.05 ng/uL plasmid from the Transformation Efficiency Kit (note: 50 pg/µL = 0.05 ng/µL) into 100 µL of competent cells. You added 900 µL SOC to your cells to get a total reaction volume of 1000 µL and then plated 100 µLs of the transformation. The plate has 250 colonies on it the next day.

      ng of DNA plated calculation: 1 µL x 0.05 ng/µL x (100 µL plated / 1000 µL total reaction volume) = 0.005 ng DNA plated

      Efficiency calculation: (250 transformants / 0.005 ng) X 1000 ng/µg = 5 x 107 CFU/µg DNA

      You can also calculate this online through the science gateway Transformation Efficiency Calculator

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.


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 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).

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 is unclear. Emails will come from traci at igem dot org if I need to follow-up with you.

Thanks very much for participating!