Difference between revisions of "Resources/Troubleshooting/Transformations"

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<li>Transform your cells with a known quantity of plasmid. iGEM provides each team with a <a href="http://parts.igem.org/Help:2017_Competent_Cell_Test_Kit">Competent Cell Test Kit and Protocol</a> for this exact purpose. We recommend you follow this protocol to transform the parts from our Competent Cell Test Kit to calculate your transformation efficiency. However, you can also use your own plasmid if you know the concentration of the DNA.  
 
<li>Transform your cells with a known quantity of plasmid. iGEM provides each team with a <a href="http://parts.igem.org/Help:2017_Competent_Cell_Test_Kit">Competent Cell Test Kit and Protocol</a> for this exact purpose. We recommend you follow this protocol to transform the parts from our Competent Cell Test Kit to calculate your transformation efficiency. However, you can also use your own plasmid if you know the concentration of the DNA.  
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<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.</li>
 
<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.</li>
  
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<li> Calculate your transformation efficiency with the following equation (CFU is colony forming units): </li>
 
<li> Calculate your transformation efficiency with the following equation (CFU is colony forming units): </li>
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<center><i>(# colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA</i></center>
 
<center><i>(# colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA</i></center>
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<ul><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:</ul>
 
<ul><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:</ul>
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Revision as of 17:21, 11 July 2017

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Transformation Troubleshooting

One of the most common problems that researchers have in the lab is transforming their DNA successfully into E. coli cells. Have you done transformations but have failed to see good results? You're not alone! Here are some tips to help you troubleshoot this problem:



Few or No Colonies

How competent are your cells? If you don't know the answer to this question, you need to run a positive transformation 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. This should be the first experiment you run for any new batch of competent cells. Never assume your cells will work correctly.

  • Transform your cells with a known quantity of plasmid. iGEM provides each team with a Competent Cell Test Kit and Protocol for this exact purpose. We recommend you follow this protocol to transform the parts from our Competent Cell Test Kit to calculate your transformation efficiency. However, 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.

  • Calculate your transformation efficiency with the following equation (CFU is colony forming units):

    • (# colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA

    • 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 in ng/µ x (volume plated / total reaction volume)

Competent Cell Test Kit

Example: You transformed 1µL of 0.05 ng/uL plasmid from the Competent Cell Test 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 colonies / 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
(Note: The DNA concentration in this tool asks for µg/µL instead of ng/µL)
The Higher, the Better: 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 106 (Batch A) and another batch that has an efficiency of 1 x 109 (Batch B), the Batch B cells will result in more colonies when transformed with the same amount of DNA. Due to it's low efficiency, Batch A may result in no colonies at all for ligations.
General Rules for Efficiency:

  1. If your efficiency is equal to or less than 1 x 107 CFU/µg DNA, use these cells for plasmid transformations

  2. If your efficiency is greater than 1 x 107 (ideally 1 x 108 or higher), use these cells for ligation and other assembly reaction transformations
Always Test Your Cells: You should remake your competent cells until your cells are

at least 5 x 107 CFU/µg DNA

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.

No Colonies with High Efficiency Cells

What if you have a high efficiency (above 5 x 107 CFU/µg DNA) but still aren't seeing many colonies? There are a handful of common mistakes that can happen during the transformation process.
  1. Incorrect antibiotic: 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.
      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.

  2. Incorrect concentration of antibiotic: Make sure you use the correct amount of antibiotic in your plates. See Antibiotic stocks for iGEM's guidelines for the correct concentration of antibiotic to use for the BioBricks plasmids.

  3. Excessive freeze-thaw: 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.

Too Many Colonies or a Lawn of Growth

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.

Plasmid transformed into highly competent cells: 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. There's are easy solutions to this problem: plate less volume of cells after the recovery step in your protocol OR dilute the plasmid going into the transformation.
Adding antibiotic to hot agar: 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 present. Ideally, the agar should be cooled to around 50°C prior to the addition of antibiotic (for BioBricks, please follow our guidelines for antibiotic stocks). Plates should also be poured at 50°C to help prevent bubbles from forming in your plates.
Improperly mixed plates: 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.
Lack of antibiotic: 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 E. coli cells that did not take up your plasmid will grow.

Possible Protocol Problems

Below are some other considerations to take into account when troubleshooting your transformation protocol itself. These are the common steps in a chemically competent protocol that can be easily altered to attempt to improve your transformation results.
Low amount of DNA transformed: 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.
Heat shock: 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°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.
Recovery Time: If you're still seeing a low number of colonies, you can also try increasing the length of your recovery time for your cells. Most protocols recommend a 60-minute recovery at 37°C for E. coli cells.
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