Never assume your cells will work correctly. Transformation Efficiency Kit Transform your cells with a known quantity of plasmid. 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. The plate has colonies on it the next day. 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 10 6 Batch A and another batch that has an efficiency of 1 x 10 9 Batch B , the Batch B cells with the higher efficiency number will result in more colonies when transformed with the same amount of DNA. This efficiency is fine for doing plasmid transformations, but it's on the low side for transforming ligation reactions or other assembly reactions. Supercoiled DNA will enter cells more easily and thus result in more colonies on your plates.
If your efficiency is greater than 5 x 10 7 ideally 1 x 10 8 or higher , use these cells for ligation and other assembly reaction transformations.
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. 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 is most often entered as nanograms or micrograms.
You can set which scale you want to work in example: nanograms or micrograms and it will give you a series of different ratio outputs you can try to optimize your ligation if the ratio fails for you. Too many colonies Sometimes with ligation reactions you can end up with a lawn of bacterial growth where it's impossible to select a single colony. While you may think this means your reaction worked really well, it actually indicates a problem with your restriction digest.
No antibiotic: The most common cause of a lawn of bacteria after ligation is plating the transformation on an agar plate with no antibiotic added. This will allow any cells, with or without your ligation product, to grow up overnight on the plate. Uncut backbone: Another possible cause of too many colonies is from having a large amount of uncut plasmid backbone in your reaction.
This makes your transformation in essence a plasmid transformation and you get far too many colonies on your plate. Remember this is one of the controls you should run for your ligations - setup a ligation reaction with your cut backbone without ligase. This control will tell you if large amounts of uncut plasmid is the cause of your problem. Press Kit Newsletter. Requirements Calendar Meetups Giant Jamboree. About Contact Sponsor Registry igem.
Colonies will give you an idea of the background due to uncut vector due to inefficient restriction digest of the backbone.
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