Help:Protocols/Transformation/Troubleshooting


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 Transformation Efficiency Kit and Protocol 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. 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.

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.

Low Efficiency

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 with the higher efficiency number will result in more colonies when transformed with the same amount of DNA.

In the example calculation above, the transformation efficiency was 5 x 107 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.

A good rule to follow is this if your efficiency is equal to or less than 5 x 107 CFU/µg DNA, use these cells for plasmid transformations. If your efficiency is greater than 5 x 107 (ideally 1 x 108 or higher), use these cells for ligation and other assembly reaction transformations.

My best advice would be to remake your competent cells until your cells are higher than 1 x 108 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.

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.

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.

Incorrect ligation or assembly reaction

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.

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.

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 going into cells with efficiencies above 5 x 108 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.

Adding antibiotic to hot agar

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 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.