DIY Electrocompetent E. coli
If you buy competent E.coli regularly, you’ll know that they are pretty expensive.
So the cost of screwing up a cloning or transformation experiment is pretty high in terms of money, as well as your time and sanity!
But you don’t need this extra worry because despite what their high commercial cost would suggest, making good quality competent E.coli is very easy. One morning’s work (with a bit of work ahead of time) is all it takes to make great electrocompetent E.coli prep.
In this article, I’ll describe a protocol for making electrocompetent E.coli that contains a variety of tricks and tweaks that make it possible to routinely get competencies of 1×10^10, with a little practice.
I’ll also describe a couple of quality control checks that you can do to validate each prep you make.
This protocol is for making a fairly large batch of cells but can be scaled down easily without loss of quality.
The tips and tweaks are as follows:
1. Keep everything fresh and chilled at all times
2. Wash the cells extensively in glycerol
3. Start with a high volume of cells so that the final competent cell aliquots are very concentrated.
4 Hand-wash the glassware before autoclaving to ensure that no detergent is present
These are all included in this protocol and the original references are listed at the bottom of this article.
The Protocol
1. Streak the strain you wish to make competent onto an LB plate and incubate overnight at the appropriate temperature.
2. The next afternoon, pick a single colony into 10 mL of LB in a sterile bottle and grow overnight in a shaking incubator at 37degC.
At this point chill the following in the freezer:
-falcon tubes or centrifuge pots (see step 5)
-1L of sterile 10% glycerol
-35 sterile cryovials, labelled with the strain name
3. In the morning, inoculate 800 mL of LB in a 2L baffled flask with 8 mL of the overnight culture and grow at 37degC in a shaking incubator.
4. Grow the culture to an OD of between 0.7 and 1.0 at 37degC. This should take around 2-3 hours.
FROM NOW ON KEEP EVERYTHING ON ICE AT ALL TIMES:
5. Transfer 400mL of the culture to 8x pre-chilled 50 mL falcon tubes (or a suitably sized sterile centrifuge pot). Chill the tubes, and the remaining 400 mL on ice for 1 hour.
6. Centrifuge for 10 minutes at 4500 rpm and 4degC then very carefully remove the supernatant.
7. Pour the remaining 400mL of culture into the tubes and repeat step 6.
8. Add 5-10 mL of chilled 10% glycerol to each falcon tube and gently re-suspend the cells. Then make up the volume in each tube to 25 mL with 10% glycerol.
9. Centrifuge for 10 minutes at 4500 rpm and 4degC then remove the supernatant.
10. Repeat steps 8 and 9 twice times. On the final repeat, pool all of the cells into 1 Falcon tube, centrifuge as before then resuspend in a final volume of 6 mL in 10% glycerol.
11. Leave the cells on ice for 10 minutes then pipette 180 ul into each cryovial and transfer immediately to the -80degC freezer.
12. Keep the remaining cell suspension for quality control checks.
Quality control checks
A batch of competent cells like this is only good if you actually know how good they are so it is worth performing a couple of simple quality control checks.
1. Phage check
Streak 35ul of cell suspension onto an LB plate and grow overnight at 37degC. If there is no phage contamination, the cells will grow to form a thick, healthy lawn.
But if phage is present, circular clearings will appear or, if there is a very high amount of phage, there will be no visible growth at all.
2. Competency check
Transform 2x 50 ul of the cell suspension with 1ul of an empty plasmid (preferably pUC18) at 0.1ng/ul. Plate 5 and 50 ul on separate plates with the appropriate antibiotic selection and grow overnight.
Count the number of colonies on the plates and calculate the number of colonies formed per ug of DNA. (e.g. If you obtain 50 colonies on the 5ul plate, the efficiency is 1×10^8).
Normally, 1×10^8 to 1×10^10 cfu/ug DNA for standard 3-5kb plasmids should be easily achievable with this protocol.
Any questions/comments? Click on the link below to discuss this article in the Bitesize Bio Bistro.
References
- Dower, W. J., Miller, J. F., and Ragsdale, C. W. (1988) High efficiency transformation of E. coli by voltage electroporation. Nucleic Acids Research, 16, 6127-6145.
- Chuang, S. E., Chen, A. L., and Chao, C. C. (1995) Growth of E. coli at low temperature dramatically increase transformation frequency by electroporation. Nucleic Acids Research, 23(9), 1641.
- Sheng, Y., Mancino, V., and Birren, B. (1995) Transformation of Escherichia coli with large DNA molecules by electroporation. Nucleic Acids Research, 23(1), 1990-1996.
- Engberg J., Andersen P. S., Nielsen L. K., Dziegiel M., Johansen L. K., Albrechtsen B., (1996) Phage display libraries of murine and human antibody fragments. Molecular Biotechnology, 6, 287-310
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Nick Oswald
Nick is a molecular biologist-turned-publisher. After a PhD in Developmental Biology and an eclectic seven years in biotech he is now Editorial Manager of Neuroendocrinology and the founder and Editor-In-Chief of Bitesize Bio. You are welcome to connect with Nick on LinkedIn
Thanks for the procedure. I’ve used this type of procedure before. I can confirm that it works very well.
We would sometimes forget to make cell stocks and run out. If we were really desperate for electrocompetent for a subcloning, we used to actually scrape some cells from a pretty new LB plate into 1mL of cold 10% glycerol. Pellet the cells and then resuspend the cells into 100uL of the cold 10% glycerol. It gives instant electrocompetent cells. Surprisingly it works!
[...] on pinpointing ligation problems (which provides some more details on ligation controls) and making competent E.coli (which includes a section on cell quality [...]
Found the article very useful (as always on this website
);I’d like to ask why are cold conditions so necessary? My teacher says that is to reduce stress on the cells,but stress in what sense?
Hi Monisha,
The cold conditions are needed because the electroporation process generates heat.
You can do electroporation without chilling the cells – I have done this in the past when I just want a couple of clones carrying my intact plasmid – but the transformation efficiency is extremely low because many of the cells get fried.
The ice just stops the cells heating up…
What if i am using the classic calcium chloride method?
Hi Monisha,
My impression of the need for cold conditions in CaCl2 transformation is that the DNA is physically swept into the cells by the “thermal current” that comes from the heat shock so the 0degC to 42degC gives more of a “sweep” and 21degC to 42degC. I am not 100% sure of this though. (see http://bitesizebio.com/2007/09/18/ecoli-electroporation-vs-chemical-transformation/)
Other possibilities:
1. The cold temperatures protect the cells from osmotic stress
2. The cells are vulnerable to lysis because of the pores CaCl2 forms in the membrane. Low temperatures may help prevent lysis.
Sorry I can’t give a definite answer but these would be my best guesses…
Unfortunately, I didn’t have much luck with this. Not sure where I went wrong, although I did have to wash them in a slightly smaller volume of 10% glycerol. Is the OD600 at which you harvest the cells particularly important, as I notice a lot of other protocols recommend 0.4-0.5?
Hi Lena,
When you say you didn’t have much luck – what do you mean? Did you get some transformants but not as much as you thought or did you get no transformants?
I beleive that the optimal OD depends on the cell line, but you should still get reasonably good results with this protocol even outside the optimal range
Nick
I got one transformant for one cell line when I plated out 50ul (and none for the other
). I’m going to try and use them today anyway, and plate out all of the electroporated cells. One minor question about the protocol, what size rotor did you use?