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EXPERIMENT #1: Transformation of Bacteria from
Antibiotic-Sensitive to Antibiotic-Resistant
Characteristic.
Each group will have the following reagents:
-Bucket of Ice containing the following microcentrifuge tubes:
a. 1x P tube with 5ul (~5ng) of pUC18 DNA that contains the ampR gene.
b. 1x W tube with 5ul water. This is your negative control (i.e. no DNA)
c. 2x C tubes, each with 480ul of SOC liquid media for growing bacteria.
d. Two tubes, each with 20ul competent E. coli host cells.
One will be labelled “+” which will receive the DNA.
One will be labelled “-” and will receive the water control.
-
agar plates (x2) contain Luria Broth (LB).
agar plates (x2) contain Luria Broth +ampicillin. (LB/Amp).
Procedure:
FIRST, A COUPLE THINGS TO KEEP IN MIND…
- Wear gloves. Although the bacteria are not pathogenic, it is good practice to treat live
cultures with care. Furthermore, the gloves also serve to protect your DNA samples.
- Competent cells are delicate. Be as gentle as possible.
1. Allow the frozen competent E. coli (labelled + and -) cells to
thaw on ice for about 15min.
2. First you will add your DNA sample to some competent
cells. Add all 5.0ul of the pUC18 DNA to one of the tubes
containing E. coli competent cells. (Pipette liquid from tube P
and put it into tube +).
3. Next you will perform a negative control experiment by just
adding water to some competent cells. Add all 5.0ul of the
water to the other tube containing E. coli competent cells.
(Pipette liquid from tube W and put it into tube -).
4. Incubate your competent cell samples on ice for about
30min. (Place + and - tube, now containing samples from P or
W, on ice).
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5. After the ice incubation, Heat shock the + and - samples at 42oC for exactly 60sec.
Immediately put tubes back on ice for a minimum of 2 minutes.
6. You will now add 480 µL of SOC media to each of your heat shocked samples. There
are two tubes of SOC media (labelled C), one for each of your samples. (Pipette 480 µL
of tube C into tube +, and pipette 480 µL of the other tube C and put into tube -).
7. Incubate tubes at 37oC for a minimum of 1hr.
8. You are now ready for spreading your competent bacterial cells onto
LB agar plates. Essentially the protocol for each plate is as follows:
- Give E. coli sample a brief mix by flicking the tube with your finger.
- Pipette 50 µL of bacterial cell culture into the center of an agar plate.
- Immediately spread the aliquot around using the spreader or “hockey stick.”
- Replace lid on plate.
Use this procedure for the following plates:
9. Incubate plates in an inverted position in
the 370C incubator overnight.
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TRANSFORMATION LAB: some notes.
This lab is actually pretty straight forward and can be done with minimal equipment.
However, you will need to provide a 42C heat source (although it only needs to maintain
this heat for ~90sec), and a 37C incubator would help.
This lab is all about getting DNA into the bacteria itself (in this case it is a commercially
available E. coli strain). And really, nobody knows what’s going on exactly…
Summarize below in a figure is the general layout of what people think is going on:
Your competent cells (meaning that your cells are primed to take up DNA) have been
pre-treated with CaCl2. Generally, it is thought that treating your E. coli bugs with CaCl2
will batter the membranes and essentially make the bacteria very unhappy. However, a
happy consequence of all this, is that the cells are now believed to contain gaping holes in
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the membrane which allows them to take up DNA. These cells are now classed as
“competent” and are very delicate. So delicate that thawing them inappropriately, or
even mixing them too vigorously could lead to their untimely deaths.
Normally, they are stored at -70C (very cold) and you should make it a point to use them
as quickly as possible after removal from the -70C fridge. Fortunately, if they were
packed in a healthy portion of dry ice they should be fine until the dry ice has evaporated
- this will probably take at least 48-72hrs (dry ice sublimes at -78.5C at 1atm or 760mm).
If you are uncertain about the health of the cells, then pipette a greater amount of
transformed bugs than outlined in the text (step 8: try 200ul instead of 50ul, or try both
amounts).
You’ll notice there is an ice incubation step for about 30minutes. During this time it is
thought that you are giving the DNA a chance to stick to the cells. This way, they have
already made contact with the cells are ready to “go in.” How it “goes in” seems to be
dependant on the heat shock step. Here, the elevated temperature probably causes your
membrane to move around (membranes actually move like fluids in a two-dimensional
plain),.. perhaps the holes get bigger, perhaps the DNA falls into the cell. Again, it’s not
quite clear how it all works. Because this step is so important, various procedures put a
lot of emphasis of making sure you adhere exactly to the heat shock time (in this case,
it is 60sec). You probably don’t have to fret about being super precise, but hopefully you
will appreciate that this whole step is probably making the cells even more unhappy so
you should try not to be too absent minded and accidentally let your heat shock go for
>2min.
The last steps can be thought of as a recuperation period. You will add 480ul of SOC
media which is simply a growth media that makes the cells happy! You will also
incubate these cells for about an hour (you can probably go as long as 2-3hrs if needed) at
37C. Again, all designed to get the cells healthy. Furthermore, the incubation period will
allow the replication of the plasmid DNA (if it got in). This will enable the transformed
bug to exhibit ampicillin resistance so that it will grow on your selective media (amp+
plates).
*notes for if you notice that your colonies are blue: In some cases we will give you
plates that have been supplemented with an additional chemical called X-gal. If this is
the case, refer to the accompanying literature provided. If you have any questions please
don’t hesitate to ask.
With the plating procedure, it’s important to make sure of the following:
- Make sure your hockey stick is sterile! Use it fresh or dip it in ethanol / flame to remove
excess ethanol.
- Make sure your hockey stick is not too hot. Count a good 10 to 15 mississippi’s before
spreading.
- DO NOT dip a flaming hockey stick into the ethanol bath!!!
- Don’t forget to mix your cell sample before you take out a sample (this is a very
common mistake)!!!
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- You want to spread your sample until you can “feel” the liquid absorbed into the plate.
You should notice a change in the friction. For 50ul, this probably won’t take very long
and you can speed things up by prewarming your plates at 37C an 1hr before spreading.
FINALLY, if you don’t have access to a 37C incubator, you can incubate your plates at
room temperature and still get the same results. You just may have to wait 2 to 3 days
instead of overnight.
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Transformation- probably a process used by bacteria
through time.
Selecting transgenic cells
Antibiotic resistance
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Plasmid vector contains an ampicillin resistance
gene making the cell resistant.
Growth of transformed cells (cells receiving the
plasmid) can be identified on agar medium
containing (e.g.) ampicillin.
Plasmids, Molecular Vectors
Plasmid
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Extrachromosomal DNA in a bacterial cell which
can replicate independently but which cannot
integrate into the host chromosome.
Drug resistance plasmids are not essential for the
cell's growth, but confer antibiotic resistance.
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Plasmids used for molecular cloning have been
artificially created by recombining fragments of
various existing plasmids.
Plasmids contain multiple cloning sites with several
restriction endonuclease sites.
Examples of Plasmids
pBR322
pUC19
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pUC19 with Insert: Note the insert disrupts the gene
coding for b-galactosidase.
Bacteria Can Get Genes from Naked DNA: Transformation

Sometimes naked DNA is taken up by bacterial cells
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This is how the transforming principle DNA got into the pneumonia bacteria in
Griffith's experiments
Some bacteria have specialized membrane proteins to bring DNA into their cells
Ca stimulates uptake of DNA into bacteria
Plasmids Allow Bacteria to Share Genes Rapidly
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Plasmids are small circular accessory "chromosomes"
Not necessary for life, but give special properties
Duplicate independently of bacterial chromosome
Some can insert into the bacterial chromosome
o Episome can reproduce when inserted or when free
Examples:
o F plasmid: makes bacterium "male"
o R plasmid: gives antibiotic resistance
Plasmids are like viruses, but have no etracellular phase