I once had the terrible experience of not being able to run an assay because I ran out of commercial stock of transformation-competent Escherichia coli (E.coli). From that day, I learned to make my own chemically-competent cells in the lab.
I recommend that everyone makes their own stash of transformation-competent E.coli stocks—among other suggested laboratory activities. First, every molecular biologist should learn how to do this. Second, it is very straightforward. Third, it will save you money and prevent emergency situations.
Today, I will show you how to make a DIY stock of chemically-competent E. coli., the workhorse in the molecular biology laboratory.
Things You Will Need
Fresh overnight culture of any E. coli strain grown in RB (rich broth)
Sterile centrifuge tubes (i.e., for Beckman JA-17 rotor)
A Spectrometer for reading the density of the E. coli
Ice-cold CaCl2 at 30 mM
5 mL micro-centrifuge tubes
Preparation of the Cells
How do you make E.coli happily take in foreign plasmids? You see, these little bugs do not normally just gobble up any foreign substances (plasmids included) for no reason. The trick is to disrupt (activate) the cell membrane of the E.coli, so that it will be ready to take in the plasmid. Chemically-competent E.coli basically means that the bacteria are pre-treated with chemicals to enable the bug to take up the plasmid when the situation requires (your experiment).
In brief, you need to grow your batch of E.coli from a small volume and expand them. Then, collect the E.coli when they are actively dividing (logarithmic growth). Because the population is in synchrony during logarithmic growth, the E.coli will be best prepared to become transformation competent. This is actually a very crucial step. In addition, when making a batch of chemically-competent E.coli, do not add any antibiotics to the growth medium. You are not amplifying plasmids, which gives the lab E.coli the antibiotic resistance. After growth, treat the little bugs with a series of cold salt buffer washes to render the membrane semi-permeable to plasmid DNA.
Here is a simple protocol on how to prepare your own chemically-competent E.coli stock.
How to Make Chemically-Competent Cells
Grow E. coli overnight on a shaker (Tip #1: no antibiotics)
Take the strain of E.coli you wish to make chemically-competent from either a glycerol stock or a freshly-streaked agar plate and inoculate it into a flask containing approximately 50 mL of RB. Let it grow overnight. As I have said, no antibiotics needed in this step!
The next day, take a small volume of the overnight E.coli culture (~500 μL) and sub-culture it into another incubation flask containing 50 mL of RB.
Grow to the right optical density (OD)
Check the OD600 every 2 to 3 hours until it reaches the desired value. Each laboratory has different OD600 values, and it ranges from 0.3 to 0.5. E.coli cultures in this OD600 range are still in the logarithmic growth phase. Once the culture reaches an OD above this range, the bacterial culture will be in the stationary phase.
Centrifuge at 8000 rpm for 5 min in sterile JA-17 tubes
After this step put everything on ice
Re-suspend in 5 mL of ice-cold CaCl2
Re-distribute into pre-chilled 1.5 mL ultra-centrifuge tubes
Spin in micro-centrifuge and pellet
Re-suspend in 500 µL ice-cold CaCl2
Aliquot 50 µL into sterile micro-centrifuge tubes
Store the tubes at -80°C freezer
Check the transformation efficiency of your chemically-competent cells by transforming with a plasmid that contains a positive selection marker
I really like the DIY movement in preparing lab reagents. We are fortunate to be doing science in this era, because there are so many “kits” available. However, it is always good to learn how to prepare a lot of reagents by yourself. These secret “personal stocks” pay huge dividends when you are in emergency situations. Not to mention that DIY is always a budget-friendly option amid periods of financial austerity. You can also try your hand at making your own electrocompetent cells.
No matter how we make measurements, there will be variation (a spread of data). Take 100 people and ask them to guess your age and you will get a range of results: some will be too low (excellent!), some too high (not so good!). It is the same with any of our laboratory experiments – […]
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