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Using Ampicillin in Plasmid DNA Isolation: What You Need to Know

Posted by The Protein Man on May 15, 2013 5:00:00 AM
The Protein Man

Question:

What you need to know about Ampicillin and Plasmid DNA Isolation?  

The Protein Man Says:

While ampicillin is commonly used as a selection marker for E. coli and other bacteria during plasmid DNA isolation, protein expression and gene cloning, there are several problems that you may encounter if you are not aware of its limitations.What are these limitations and how can you avoid them? Here are some things that you definitely need to know.

describe the imageUsing Ampicillin as a Selection Marker for Plasmid DNA Isolation

There are several selectable markers that can be used to identify bacterial cells that contain a specific trait. Most of these markers are genes that confer resistance to antibiotics such as ampicillin, kanamycin, tetracycline and chloramphenicol.

By introducing a selectable marker gene into the bacterial cells, the colonies that have successfully taken up the plasmid will most likely develop a resistance against that particular antibiotic while those that do not would eventually perish.The surviving colonies can then be isolated, propagated and used for subsequent downstream experimentations.

Ampicillin is commonly used as a selection marker since it binds to and inhibits the action of several enzymes that are involved in the synthesis of the cell wall. The ampicillin-resistant gene (ampR), on the other hand, catalyzes the hydrolysis of the B-lactam ring of ampicillin and naturally detoxifies the drug.

However, if the bacterial culture is not handled properly, there is a possibility that the ampR cellswillsecreteenough beta-lactamase that can inactivate the ampicillin in the culture. When this happens, you will get poor yielding plasmid preps and gene expression in your liquid cultures and satellite colony formation on your transformation plates. To avoid such things from taking place, here are some tips that you may need to consider:

  • Avoid over-saturation. For best results, don't grow bacterial cultures with a density higher than OD600=3 and don't leave your cultures for more than 8 to 10 hours.

  • Always use fresh stocks or plates. Old stocks and plates may have reduced ampicillin concentration.

  • Use a higher concentration. This will make it impossible for beta-lactamase to inactivate all of the ampicillin in your culture.

  • Take special precaution when preparing your starter culture. Remove all traces of beta-lactamase from the medium by pelleting and resuspending your starter culture in fresh, antibiotic-free medium prior to inoculating the main culture.

  • Make the switch. If everything else fails, use carbenicillin.It is not immune to the action of beta-lactamase but it will take a longer time to inactivate it. However, please take note that this antibiotic is more expensive than ampicillin.

Topics: Molecular Biology

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