How Long Do Competent Cells Last?

by Simon Currie

Competent cells such as DH5a, DH10B, and BL21 will maintain their transformation efficiency for at least a year with proper storage. It is important to store competent cells in the -80 ºC freezer and avoid repeat freeze-thaw cycles to preserve their competency for as long as possible.  

You reach into the deep, recesses of your -80 ºC freezer. After some searching, you find it: the competent cells you need. The box of competent cells is frosted over and it looks like it’s been in the freezer for a while. Naturally, you wonder: “are these cells any good? Will they work for the experiment that I’m about to do?”

Competent cells such as DH5a, DH10B, and BL21 will maintain their transformation efficiency for at least a year with proper storage. It is important to store competent cells in the -80 ºC freezer and avoid repeat freeze-thaw cycles to preserve their competency for as long as possible.  

In this article, we’ll cover proper storage techniques to maintain your competent cells’ transformation efficiency for as long as possible, and we’ll discuss what to consider if your cells are over one year old.

Article Table of Contents

How to properly store competent cells

Store in a -80 freezer

Single-use aliquots

Use your competent cells immediately after thawing them

Will my competent cells work after one year?

How to properly store competent cells

As we mentioned, competent cells should maintain their transformational efficiency with proper storage. So, what exactly is proper storage?

There are three key factors when properly storing competent cells: store long-term in a -80 ºC freezer, store in single-use aliquots to avoid repeat freeze-thaw cycles, and use them immediately after thawing.

·         For long-term storage, store competent cells in a -80 ºC freezer

·         Store competent cells in single-use aliquots and avoid repeat freeze-thaw cycles

·         After thawing competent cells, use them immediately, do not store on ice for days

Storing competent cells in a -80 ºC freezer

For long-term storage, it is critical to keep your competent cells in a -80 ºC freezer. At this temperature they will maintain their competency for at least one year.

You might be thinking, “frozen is frozen, what difference does it make if I use a -20 ºC or -80 ºC freezer?” In this case it actually makes a big difference. When stored in the -20 ºC freezer, competent cells lose more than 90% of their transformation efficiency within a few days (Williams, 2015).

Why does -80 ºC keep cells competent for a year or longer compared to days at -20 ºC? While samples at both temperatures appear frozen to the unaided eye, there is actually a big difference in the molecular motions that occur at the two temperatures. Storage at -80 ºC drastically slows down molecular movements, which delays reactions that degrade biological samples, thereby better preserving samples. In the case of competent cells, maintaining the membrane that confers competency is the key for maintaining transformation efficiency, and this form is maintained much longer at -80 ºC.

Single-use aliquots

The efficiency of competent cells drops roughly twofold each time they go through a freeze-thaw cycle (Protocol Online, 2007).

A freeze-thaw cycle means each time that you open the -80 ºC freezer, thaw your cells on ice, and then refreeze all or some portion of those cells and put them back in the -80 ºC freezer.

So, as a best-practice, you’ll want to avoid having your competent cells endure any freeze-thaw cycles before you’re ready to use them.

We recommend using 25 microliters of competent cells for each transformation reaction. GoldBio sells competent cells in 50 microliter aliquots, which is convenient because you almost always want to include at least one control alongside your actual transformation reaction. This means that the 50 microliters of competent cells in each vial is effectively a single-use aliquot that can be split into two 25-microliter aliquots for your transformation and one control reaction.

When you’re doing your transformation, you can pull out exactly however many competent cell aliquots you plan on using, and leave the rest frozen until they’re needed (Figure 1).

Figure 1. Repeated freeze-thaw cycles drop transformation efficiency (top). Aliquoting competent cells into single-use volumes minimizes freeze-thaw cycles and maximizes transformation efficiency (bottom).

Keep in mind the act of aliquoting involves thawing your cells and refreezing them. Therefore, this step means you will be introducing one freeze-thaw cycle, which will lead to a reduction in transformation efficiency. However, by having aliquots available, you are protecting your cells from repeated freeze-thaw cycles that will reduce efficiency each time.

Use your competent cells immediately after thawing them

Ok, now that you’re ready to use your single-use aliquots, you pull them out of the freezer and place them in an ice bucket to thaw. After they’ve thawed for a few minutes, you’ll want to start the transformation protocol immediately to get the highest transformation efficiency.

That’s the ideal scenario, but I know unexpected things come up in lab all the time. For example, your professor stops by and says that she needs that important data you’ve been working on now for her presentation tomorrow. So, what will happen if your competent cells sit on ice for a little bit before you use them?

If it’s only been a few hours, then the transformation efficiency is probably still good enough, so I would go ahead and use them. But, if those cells have been in your ice bucket for a few days, toss them and grab a fresh aliquot out of the -80 ºC freezer. It’s probably not worth wasting your time for poor transformation efficiency.

Will my competent cells work after one year?

What if you have competent cells that have been in the freezer for longer than a year? Is it even worth trying to use them? For this thought experiment, I’m assuming that you have followed all of the above best practices, meaning these cells have been stored in the -80 ºC freezer, they haven’t gone through multiple freeze-thaw cycles, and they haven’t been thawed for extended periods of time.

So, in this scenario, is it even worth trying to use competent cells that have been in your -80 ºC freezer for over a year? I personally think it’s worth trying those cells out, with the caveat that I would only use them for standard procedures where transformation efficiency is not crucial.

Scientists have reported successfully using competent cells that were up to three years old (Tahir, 2019). In my own experience, I’ve definitely had success with competent cells that are over a year old. To be honest, we weren’t being that careful about how old the cells were. So, I can say that I’ve successfully used competent cells that were over a year old, but less than the length of my Ph.D. studies (Please, don’t ask how long that too.).

When using questionable competent cells, I recommend using these on techniques where transformation efficiency is not that important. For example, when you’re cloning a single plasmid with DH5a or DH10B cells, or transforming BL21 cells for protein expression, you really only need to get one colony, so your transformation efficiency can be low and the experiment can still be a success (Figure 2).

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competent cells Competent CellsDh5 alpha electrocompetent cells Shelf Life Simon Currie

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