How to Make Chemically Competent E. coli
Summary
TLDRIn this video on synthetic biology, the process of preparing competent E. coli cells is demonstrated. The steps include preparing two calcium chloride solutions, inoculating and growing E. coli, and diluting the culture for optimal competency. After centrifugation, cells are resuspended in calcium chloride and glycerol solutions and kept cold to maintain competency. The cells are then aliquoted into smaller tubes for immediate use or storage. Careful temperature control is emphasized throughout the procedure to ensure cell viability and efficiency during transformation.
Takeaways
- 🧪 Preparing competent cells involves making two solutions: 100 mM calcium chloride and 100 mM calcium chloride with 15% glycerol.
- 🔬 Accurate measurement is not critical for these solutions; 'close is good enough' as they are forgiving.
- ❄️ Solutions should be stored on ice to maintain a temperature of 4 degrees Celsius to prevent cell lysis.
- 🌡️ The process starts with a single colony of E.coli to ensure genetic homogeneity and lack of contamination.
- 🚫 It's important to work with log phase E.coli for competent cell preparation as they are healthier and more competent.
- 🌡️ The culture is grown at 37 degrees Celsius with shaking until it reaches an OD of 0.5 or 0.6, indicating mid-log phase.
- 🧊 After centrifugation, cells are resuspended in calcium chloride solution and kept on ice to maintain low temperatures.
- 🕒 The cells are left on ice for at least four hours, or even overnight, to become competent.
- 🔄 A second round of centrifugation is performed, and cells are resuspended in calcium chloride with glycerol for transformation readiness.
- 🏗️ The final step involves aliquoting the cells into smaller tubes for convenient use in transformations, with each tube containing enough for multiple uses.
Q & A
What are the two solutions needed to prepare competent cells in synthetic biology?
-The two solutions needed are a 100 millimolar solution of calcium chloride and another solution of 100 millimolar calcium chloride with 15% glycerol.
How much calcium chloride is required to prepare the solutions?
-One and a half grams of calcium chloride is required for both solutions.
Why is it important to label the solutions near the top and what is the risk if not done so?
-The solutions should be labeled near the top because they will be stored on ice, and if the labels get wet, they can fall off.
What is the purpose of adding water to the calcium chloride solution before the full 100 milliliters?
-Adding water before the full volume allows room for the glycerol to be added later without exceeding the total volume.
Why is it recommended to be careful when pipetting glycerol?
-Glycerol can be difficult to pipette and can coat the entire pipette if not done carefully, which can affect the accuracy of the measurement.
What is the significance of storing the prepared solutions on ice?
-Storing the solutions on ice is crucial to maintain the cells at four degrees Celsius, preventing them from lysing if the temperature rises.
What type of E. coli strain is used to start the culture for making competent cells?
-A wild type strain of E. coli, specifically mg 1655, is used, but other cloning strains like nab turbo or dh5 alpha could also be used.
Why is it important to start the culture from a single colony of E. coli?
-Starting from a single colony ensures that the cells are genetically homogeneous and not contaminated.
What is the purpose of diluting the saturated E. coli culture into a fresh 50 ml culture?
-Diluting the culture helps to use freshly growing log phase E. coli, which are the healthiest and most competent for transformation.
At what optical density (OD) should the E. coli cells be to proceed with the competent cell protocol?
-The cells should be at an OD of 0.5 or 0.6, indicating they are in the mid-log phase.
How are the cells prepared for the second round of centrifugation in the competent cell protocol?
-After the first round of centrifugation, the cells are resuspended in calcium chloride solution and left on ice for at least four hours. For the second round, they are resuspended in calcium chloride and glycerol solution.
Outlines
🔬 Preparing Solutions for Competent Cells
The video begins with the preparation of two solutions: a 100 mM calcium chloride solution and a 100 mM calcium chloride solution with 15% glycerol. The process involves weighing out 1.5 grams of calcium chloride and adding water to make up the volume to 100 milliliters for the first solution. For the second solution, water is added to 50 milliliters, and then 15 milliliters of 100% glycerol is carefully pipetted in. The solutions are mixed and stored on ice to maintain a temperature of 4 degrees Celsius, which is crucial to prevent cell lysis. Proper labeling of the solutions is emphasized, and the importance of keeping the cells at a low temperature throughout the process is highlighted.
🌡 Culturing E.coli for Competent Cells
The second paragraph details the process of culturing E.coli for making competent cells. It starts with inoculating a single colony of E.coli into 5 milliliters of LB media in a series of 14 Falcon tubes. The culture is then grown at 37 degrees Celsius with shaking overnight for 8 to 12 hours. The next day, the saturated E.coli culture is diluted 200-fold into a fresh 50 milliliter culture of LB media. The culture is grown further until it reaches an optical density (OD) of 0.5 or 0.6, indicating that the cells are in the mid-log phase and ready to be made competent. The cells are then collected by centrifugation to form a pellet, which is a precursor to the next step in the competent cell protocol.
🧊 Transforming Cells into Competent State
The final paragraph describes the transformation of pelleted E.coli cells into competent cells. After pouring off the supernatant from the centrifugation, the cells are immediately placed on ice to maintain a temperature of 4 degrees Celsius. The cells are resuspended in 15 milliliters of calcium chloride solution and gently pipetted to ensure a uniform suspension. The cells are then left on ice for at least four hours or overnight. In the subsequent round of centrifugation, the cells are resuspended in 4 milliliters of the calcium chloride and glycerol solution. The competent cells are then aliquoted into smaller Eppendorf tubes for convenience in transformation, with each tube containing 200 microliters, sufficient for multiple transformations. The competent cells are ready for immediate use or can be stored at -80 degrees Celsius indefinitely.
Mindmap
Keywords
💡Competent cells
💡Calcium chloride
💡Glycerol
💡LB media
💡E. coli
💡Transformation
💡Centrifugation
💡OD (Optical Density)
💡Incubation
💡Aliquoting
Highlights
Introduction to preparing competent cells in synthetic biology.
Preparation of 100 millimolar calcium chloride solution.
Preparation of 100 millimolar calcium chloride with 15% glycerol solution.
Labeling of solutions with care to avoid damage from ice.
Adding water to the calcium chloride solution for proper volume.
Precautions while pipetting glycerol due to its stickiness.
Mixing and storing solutions on ice for later use.
Importance of maintaining a cold temperature to prevent cell lysis.
Starting a culture of E.coli for competent cell preparation.
Use of a single colony for genetic homogeneity.
Incubation of E.coli culture at 37 degrees for optimal growth.
Dilution of saturated E.coli culture into fresh media.
Growth of E.coli in a larger flask for better aeration.
Centrifugation to pellet cells for competent cell preparation.
Resuspension of cells in calcium chloride solution.
Centrifugation and resuspension in calcium chloride with glycerol.
Aliquoting competent cells into smaller tubes for convenience.
Storage of competent cells at -80 degrees for long-term use.
Transcripts
[Music]
hi welcome back to synthetic biology 1
today we're going to be preparing
competent cells so how I like to start
is by preparing my solutions so we have
two solutions that we need to prepare
one is a hundred millimolar solution of
calcium chloride and another one is 100
millimolar calcium chloride with 15%
glycerol so we'll need one and a half
grams of calcium chloride two times
[Music]
all right and let's not forget to label
our solutions so this guy is gonna be
our 100 millimolar calcium chloride and
over here 100 millimolar calcium
chloride with 15% glycerol ok so label
these and I'm being careful to label
these bottles near the top because we're
gonna put them on ice and if the labels
get wet they can fall off ok so now I'm
gonna take my water and I'll go ahead
and add it to the calcium chloride
solution bringing the total volume of
the solution up to 100 milliliters just
pour that in top it off at 100
milliliters I'm also going to add some
water to the calcium chloride solution
but I'm not gonna add the full 100
milliliters yet I'm just going to take
it up to 50 mils so that there's room
left over for adding the glycerol okay
so now let's pipette the glycerol I need
15 mils of glycerol and for this I'm
using a hundred percent glycerol
solution and it can be really a pain to
pipette so I'll have to be extra careful
one tip here is just put the tip of the
pipette into the glycerol because
otherwise the entire pipette will be
coated with glycerol to be a little
patient to get exactly the right level
let the excess glycerol drip off
and now this is why I added some water
to the solution because when I when I
add the glycerol I want to pipette up
and down just to wash the excess
glycerol from the pipette tip
okay cool and now we'll take the water
and just top off the solution bringing
the total volume up to 100 mils you can
you can tell I'm not being too careful
with my measurements for these solutions
and that's okay I don't need to be there
they're quite forgiving and close is
good enough okay
there's my two two solutions prepared
mix them up and I'll store these on ice
so you want to store them on ice so the
most important thing about preparing
competent cells is that once we've
resuspended the cells in the calcium
chloride solution we want to keep them
always ever after at four degrees
Celsius because if we allow the
temperature to come back up the cells
might start to lyse okay so that's our
solutions now we're going to go ahead
and start the culture of cells that
we're going to actually be making
competent so for this I have a plate of
mg 165 5 you might want to use nab turbo
or dh5 alpha or some other cloning
strain this is just a wild type strain
of e.coli and it's it's what I happen to
have ready around the lab okay so we're
going to start out five mill culture of
e.coli in LB media
so these are 14 male Falcon tubes don't
forget to label
see there's my five mils and now I am
going to take an inoculation loop and
pick just a single colony of e.coli from
my plate to start my culture with it's
important when you make competent cells
to always start from a single colony of
e.coli because that way you know that
the cells are genetically homogeneous
and not contaminated okay so now I'm
going to pop this in the incubator and
grow it at 37 degrees with shaking
overnight eight to twelve hours
okay so it's eight to twelve hours later
and my culture of e.coli is nicely
saturated so the next morning when
you're ready to prepare you're competent
cells when you come in what you do is
you'll take these cells and we're going
to dilute them by a factor of 200 into a
fresh 50 milk culture so we always want
to make competent cells using freshly
growing log phase e.coli because they're
the healthiest and they'll be the most
competent so for my my 50 ml culture I'm
using a 250 mil flask it's important to
use a much larger flask than the volume
of cells that you're growing so that the
cells are nicely mixed and well aerated
so they can grow quickly
now to my 50ml of lb media I'm adding
250 microliters of my saturated e.coli
solution
okay
I mix that around so we'll grow that in
the incubator at 37 degrees for two or
three hours until the OD reaches 0.5 or
0.6 which indicates that the cells are
in the mid log phase and ready to become
competent
it's two or three hours later and I've
collected my cells from the incubator
and they're at an OD of exactly 0.5 and
so they are ready to go for the next
step of the competent cell protocol so
I'm going to take my my culture of
e.coli and my freshly log phase e coli
and I'll add it to a 50 ml Falcon tube
like this
and then put it in the centrifuge and
spin it down at a maximum speed for ten
minutes to produce the pellet of cells
for the next step in the protocol
okay so now we've collected our pelleted
cells let's turn them into competent
cells by adding calcium chloride
solution so the first thing that I'm
going to do is very quickly pour off the
supernatant from the centrifugation
store these cells immediately on ice so
from now on everything that touches
these cells should be at four degrees
Celsius or colder now we're going to
resuspend these cells in our calcium
chloride solution here 15 mils
okay so i'll resuspend the cells by
pipetting up and down the pellet should
come apart pretty easily after a few
rounds of pipetting giving us a nice
uniform resuspension of cells we don't
want to spend too long doing this or the
cells will start to warm up okay now I'm
going to leave these cells on ice for at
least four hours or it could be longer
even overnight okay so after the second
round of centrifugation we will treat
the cells the same as before except now
we are going to resuspend them in four
mils of the calcium chloride and
glycerol solution so I'll go ahead and
pour off the wastes
and resuspend in four mils of calcium
chloride glycerol
okay so now these cells are ready to use
they're ready to transform or to store
however you want for the final step I'm
going to aliquot this large culture of
cells into smaller eppendorf tubes that
will be more convenient to transform so
for that I am going to need even more
ice and I'm using this aluminum rack to
cool the cells so this will transmit the
heat very rapidly from the tubes to the
ice and keep everything nice and cold so
I like to aliquot into these 1.5 mil
eppendorf tubes and I usually do 200
microlitre a loquats so a typical
transformation will use 20 microliters
of cells so 200 microlitre a loquats is
sufficient for 10 transformations from
one tube
okay so these competent cells are
finished they are ready to use
immediately or they can be stored at
minus eighty indefinitely
[Music]
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