Difference between revisions of "Troubleshooting"

 
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                      <h4>Troubleshooting</h4>
 
 
<ul>  
 
<ul>  
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<a href=""><li class="menusubtopic"> Troubleshooting </li></a>
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                               <a href= "https://2015.igem.org/Troubleshooting#intro"> <li>Introduction</li></a>
 
                               <a href= "https://2015.igem.org/Troubleshooting#intro"> <li>Introduction</li></a>
 
                               <a href= "https://2015.igem.org/Troubleshooting#tips"> <li>General Tips and Tricks</li></a>
 
                               <a href= "https://2015.igem.org/Troubleshooting#tips"> <li>General Tips and Tricks</li></a>
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                              <a href= "https://2015.igem.org/Troubleshooting/Transformation"> <li> - Transformation</li></a>
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                              <a href="https://2015.igem.org/Troubleshooting/Restriction_Digest_and_Ligation"> <li> - Restriction Digest and Ligation</li>
 
                               <a href= "https://2015.igem.org/Troubleshooting#protocols"> <li>iGEM Protocols</li></a>
 
                               <a href= "https://2015.igem.org/Troubleshooting#protocols"> <li>iGEM Protocols</li></a>
 
                               <a href= "https://2015.igem.org/Troubleshooting#answer"> <li>Answers</li></a>
 
                               <a href= "https://2015.igem.org/Troubleshooting#answer"> <li>Answers</li></a>
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<div class="alertMessage"> Page is under construction. <br>iGEM HQ is currently working on updating this information
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for the iGEM 2015 competition. </div>
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<ul>
 
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<li>Read about <a href="#tips">General Tips and Tricks</a> </li>
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<li>Read about <a href="#tips">General Tips and Tricks</a> with a focus on <a href="https://2015.igem.org/Troubleshooting/Transformation">Transformations</a>, <a href="https://2015.igem.org/Troubleshooting/Restriction">Restriction Digests</a>, and <a href="https://2015.igem.org/Troubleshooting/Ligation">Ligations</a></li>
 
<li>Have a cloning problem? <a href="#ask">Ask a question!</a></li>
 
<li>Have a cloning problem? <a href="#ask">Ask a question!</a></li>
 
         <li>See the <a href="#answers">Answers to Your Questions</a> (which will be updated throughout the iGEM season!) </li>
 
         <li>See the <a href="#answers">Answers to Your Questions</a> (which will be updated throughout the iGEM season!) </li>
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<h2 id="intro">Introduction</h2>
 
<h2 id="intro">Introduction</h2>
 
<p>
 
<p>
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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!
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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!
 
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<h2 id="tips">General Tips and Tricks for Transformation Problems</h2>
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<h2 id="tips">General Tips and Tricks</h2>
 
<p>
 
<p>
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One of the most common problems that researchers have in the lab is transforming their DNA successfully into <i>E. coli</i> cells. 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:
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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.
 +
<br><br>
  
 +
<b>General Transformation Issues</b>
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<br>
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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 <i>E. coli</i> cells as the transformation host.
 
<ul>
 
<ul>
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    <li>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 transformation efficiency! This number is what researchers use to determine how competent their cells are and is an important calculation to run for every new batch of competent cells you make or purchase. <b>This should be the first experiment you run for any new batch of competent cells.</b> Never assume your cells will work correctly.
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                        <li><a href="https://2015.igem.org/Troubleshooting/Transformation"><i>E. coli</i> Transformations</a></li>
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</ul>
  
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          <ol><li>Transform your cells with a known quantity of plasmid. iGEM provides each team with a <a href="http://parts.igem.org/Help:Transformation_Efficiency_Kit">Transformation Efficiency Kit and Protocol</a> for this exact purpose. We recommend you follow this protocol to transform the parts from our Transformation Efficiency Kit to calculate your transformation efficiency. However, you can also use your own plasmid if you know the concentration of the DNA. <br><b>Important:</b> You <i>must</i> 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.
 
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<br><br>
 
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                <li> Calculate your transformation efficiency with the following equation (CFU is colony forming units):
 
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<br><i>(# colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA</i>
 
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<br><br>
 
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                  <ol><li>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:
 
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<br><i>volume of plasmid used in µL x concentration of DNA x (volume plated / total reaction volume)</i></ol>
 
 
<br>
 
<br>
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                  <li><b>Example:</b> 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.
 
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<br><br>
 
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<b>ng of DNA plated calculation:</b> 1 µL x 0.05 ng/µL x (100 µL plated / 1000 µL total reaction volume) = 0.005 ng DNA plated
 
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<br>
 
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<b>Efficiency calculation:</b> (250 colonies / 0.005 ng) X 1000 ng/µg = 5 x 10<sup>7</sup> CFU/µg DNA
 
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<br><br>You can also calculate this online through the Science Gateway <a href="http://www.sciencegateway.org/tools/transform.htm">Transformation Efficiency Calculator</a><br><br>
 
  
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</li></li></li></ol>
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<b>General BioBricks Cloning Problems</b>
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<li>So, what if you've calculated your transformation efficiency but you're still not seeing many (or any!) colonies? The answer could be that your transformation efficiency is on the low side and as a result you aren't seeing many colonies.
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<ol><li><b>Low Efficiency:</b> For transformations, it's rather simple: the higher the competency of your cells, the more colonies you'll see on your plate. Thus, if you have a batch of competent cells that have an efficiency of 1 x 10<sup>6</sup> (Batch A) and another batch that has an efficiency of 1 x 10<sup>9</sup> (Batch B), the Batch B cells with the higher efficiency number will result in more colonies when transformed with the same amount of DNA.
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<br><ol>In the example calculation above, the transformation efficiency was 5 x 10<sup>7</sup> CFU/µg DNA. This efficiency is fine for doing plasmid transformations, but it's on the low side for transforming ligation reactions or other assembly reactions. This is due to the supercoiled nature of plasmid DNA versus the non-supercoiled DNA in ligation reactions. Supercoiled DNA will enter cells more easily and thus result in more colonies on your plates.
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<br><b>A good rule to follow is this:</b> if your efficiency is equal to or less than 5 x 10<sup>7</sup> CFU/µg DNA, use these cells for plasmid transformations. If your efficiency is greater than 5 x 10<sup>7</sup> (ideally 1 x 10<sup>8</sup> or higher), use these cells for ligation and other assembly reaction transformations.
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<br>My best advice would be to remake your competent cells until your cells are <b><i>higher than 1 x 10<sup>8</sup> CFU/µg DNA</b></i> before transforming ligations or assembly reactions. This may take more time at first since making competent cells can be tricky, but it will save you a lot of time later on if you know you have a reliable batch of highly competent cells.
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</li></ol></ol>
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<br>
 
<br>
 +
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.
  
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<li>What if you have a high efficiency but still aren't seeing many colonies? There are a handful of common mistakes that can happen during the transformation process.
 
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<ol><li><b>Incorrect antibiotic:</b> Double-check that you are plating on the correct antibiotic. This is a very common (and easy) mistake that happens, especially when transforming multiple reactions at once.
 
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<ol>If you plated on the correct antibiotic but still see few or no colonies, then you may need to re-check your ligation or assembly reaction. You may have used an incorrect part in your reaction, thus resulting in an incorrect antibiotic backbone.</ol>
 
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<li><b>Incorrect concentration of antibiotic:</b> Make sure you use the correct amount of antibiotic in your plates. See <a href="http://parts.igem.org/Help:Protocols/Antibiotic_Stocks">Antibiotic stocks</a> for iGEM's guidelines for the correct concentration of antibiotic to use for the BioBricks plasmids.
 
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<li><b>Excessive freeze-thaw:</b> If you are using competent cells that were thawed, re-frozen, and thawed again for transformation, you will see a large decrease in efficiency (up to a two-fold drop in efficiency). It's best to use competent cells that have never been previously thawed for best results.
 
  
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</li></li></ol>
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<ul>
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<br>
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                        <li><a href="https://2015.igem.org/Troubleshooting/Ligation">Restriction Digest and Ligation</a></li>
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<li>Another problem you may encounter is having far too many colonies that result in a lawn of growth on your plates. Your first reaction may be to think that this is a good result, but often this result indicates a problem. Below are some common problems that can result in a lawn of bacteria growing instead of single, isolated colonies after transforming your DNA.
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<ol>
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<li><b>Plasmid transformed into highly competent cells:</b> If you have a high transformation efficiency and you transform plasmid, you can sometimes get a lawn of cells growing. This most often occurs if you have a high plasmid concentration going into cells with efficiencies above 5 x 10<sup>8</sup> CFU/µg DNA. There's an easy solution to this problem: plate fewer cells after the recovery step in your protocol. If you plated 100 µL of cells, you should plate 10-20 µL the next time you do a plasmid transformation. This will dilute the cells across the plate and should result in isolated colonies.
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<li><b>Adding antibiotic to hot agar:</b> When making plates, you need to make sure the agar is cool enough for the antibiotic to be added while still being molten. If the agar is too hot when you add the antibiotics, you can breakdown the antibiotic and thus make plates with little to no antibiotic resistance. Ideally, the agar should be cooled to around 50&deg;C prior to the addition of antibiotic (for BioBricks, please follow our guidelines for <a href="http://parts.igem.org/Help:Protocols/Antibiotic_Stocks">antibiotic stocks</a>). Plates should also be poured at 50&deg;C to help prevent bubbles from forming in your plates.
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<li><b>Improperly mixed plates:</b> If you make your plates without the aid of a stir bar to help mix the antibiotic into the agar, you can pour plates that have an uneven amount of antibiotic in them. Likewise, if you spread plate antibiotic onto your plates after they harden, you need to make sure the plate is properly covered. Patches of dense growth can result from improperly mixed or spread plated antibiotic, making it difficult to select an isolated colony.
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<li><b>Lack of antibiotic:</b> You may have forgot to add the antibiotic to your plates or you may have used a plate without antibiotic by mistake. A lawn will result if you use a non-selective plate because the <i>E. coli</i> cells that did not take up your plasmid will grow. I recommend that you mark the sides of your plates with a permanent marker (a thick Sharpie works well) with a series of lines to indicate the type of plate they are, including plates without antibiotic. This is easily done by stacking empty petri dishes, holding the top of the stack with one hand, then running a marker up the stack with your other hand. This creates a vertical line along the edge of each plate in your stack. Different colors and / or different number of lines can be used to denote different types of plates. Avoid using red and green markers as the color fades quickly.
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</li></li></ol>
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<br>
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<li>Below are some other considerations to take into account when troubleshooting your cloning problems, particularly as it relates to transformations.
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<ol>
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<li><b>Low amount of DNA transformed:</b> If you tried transforming 1-2 µL of a ligation, you may have transformed a very low amount of DNA into your cells if your reactions have low concentrations going into them. I generally transform 5 µL of ligation reactions into my competent cells to ensure enough DNA has gone into the transformation. This often helps increase the number of colonies you'll see on your plates.
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<li><b>Heat shock:</b> If you follow a chemically competent protocol, heat shocking your cells is often a part of your transformation protocol. Depending on the type of tube you use, you may need to alter your heat shock parameters. Generally, a water bath or thermocycler set to 42&deg;C will work well for heat shocking your cells. For PCR tubes and other thin-walled tubes, a shorter heat shock time of 45 seconds may be fine. For microcentrifuge tubes or other thick-walled tubes, I would recommend increasing the time to 60-90 seconds.
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<li><b>Recovery Time:</b> If you're still seeing a low number of colonies, you can also try altering the length of your recovery time for your cells. Most protocols recommend a 60-minute recovery at 37&deg;C for <i>E. coli</i> cells.
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</ul>
 
</ul>
  
 
<br>
 
<br>
 +
<b>Note:</b>If you have other tips you'd like me to include in these pages, please send them using the <a href= "https://2015.igem.org/Troubleshooting#ask">question form</a> 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 <i> traci AT igem DOT org</i>. Your feedback will help us track how useful this section is so we can improve it throughout the competition.
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<br><br>
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<h2 id="protocols">iGEM Protocols</h2>
 
<h2 id="protocols">iGEM Protocols</h2>
 
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<b>BioBrick Cloning</b>
 
<ul>
 
<ul>
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<li><a href="http://parts.igem.org/Help:3A_Assembly_Kit">3A assembly kit</a></li>
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<li><a href="http://parts.igem.org/Help:Protocols/3A_Assembly">3A Assembly</a></li>
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<li><a href="http://parts.igem.org/Help:Transformation_Efficiency_Kit">Transformation efficiency kit</a></li>
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<li><a href="http://parts.igem.org/Help:Prefix-Suffix">Prefix and Suffix</a></li>
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<li><a href="http://parts.igem.org/Help:Protocols/PCR_Standardization">PCR standardization</a></li>
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                        <li><a href="http://parts.igem.org/Help:Protocols/PCR_Standardization">PCR standardization</a></li>
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<li><a href="http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones">Linearized plasmid backbone</li>
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<li><a href="http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones">Linearized plasmid backbone</a></li>
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<li><a href="http://parts.igem.org/Help:Protocols/Transformation">Transformation</a></li>
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<li><a href="http://parts.igem.org/Help:Protocols/Miniprep">Miniprep</a></li>
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<li><a href="http://parts.igem.org/Help:Protocols/Restriction_Digest">Restriction digest</a></li>
 
<li><a href="http://parts.igem.org/Help:Protocols/Restriction_Digest">Restriction digest</a></li>
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<li><a href="http://parts.igem.org/Help:Protocols/Ligation">Ligation</a></li>
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 +
</ul>
 +
<br>
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<b>Cloning protocols that can be applied to BioBricks and non-BioBricks assemblies</b>
 +
<ul>
 
<li><a href="http://parts.igem.org/Help:Protocols/Competent_Cells">Making competent cells</a></li>
 
<li><a href="http://parts.igem.org/Help:Protocols/Competent_Cells">Making competent cells</a></li>
 
<li><a href="http://parts.igem.org/Help:Protocols/Antibiotic_Stocks">Antibiotic stocks</a></li>
 
<li><a href="http://parts.igem.org/Help:Protocols/Antibiotic_Stocks">Antibiotic stocks</a></li>
 +
                        <li><a href="http://parts.igem.org/Help:Protocols/Miniprep">Miniprep</a></li>
 +
<li><a href="http://parts.igem.org/Help:Protocols/Ligation">Ligation</a></li>
 +
                        <li><a href="http://parts.igem.org/Help:Protocols/Transformation">Transformation</a></li>
 +
<li><a href="http://parts.igem.org/Help:Transformation_Efficiency_Kit">Transformation efficiency kit</a></li>
 +
 
</ul>
 
</ul>
 
<br>
 
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<h2 id="ask">Ask Your Questions Here!</h2>
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<h2 id="ask">Ask Your Questions and Submit Comments Here!</h2>
 
<p>
 
<p>
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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).
+
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).  
 +
 
 
<br><br>
 
<br><br>
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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 <i>traci at igem dot org</i> if I need to follow-up with you.
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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-<i>E. coli</i> chassis!
 +
 
 +
<br><br>
 +
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!
 +
 
 +
<br><br>
 +
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 <i>traci at igem dot org</i> if I need to follow-up with you.
 
<br><br>
 
<br><br>
 
Thanks very much for participating!
 
Thanks very much for participating!
 +
<br><br>
 +
<b>Important:</b> If you have trouble using the form below, please send me an email at <i>traci AT igem DOT org</i> 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.
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</p>
 
</p>
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<center>
 
<center>

Latest revision as of 21:28, 23 July 2015

Learn about cloning troubleshooting here!

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 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.

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


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

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.