Lab 5: Cloning PCR products
Objective:
In this lab, we will clone fresh 16S rRNA products amplified by PCR. Cloning is an
essential tool in molecular biology for many reasons. Here I’ve listed a few of these
reasons relevant to our experiments:
1. DNA is not very stable and will degrade over time. Thus, there needs to be a way
to archive your molecular biology projects. Cloning allows for the archiving of
your experiments so that you can return to them in the future if needed.
2. In the PCR lab, you amplified community 16S rRNA genes. These PCR products
are all mixed together in one tube and it is your job to “separate” them from one
another. Cloning allows for the separation of mixed products so the information
contained in your PCR products can be analyzed.
The following diagram shows the steps in the cloning process:
Step 1: DNA extraction
Extracted “community” DNA
We start out with extracted DNA. In that solution we have DNA from many different
bacteria (among other things). In the above figure, image the different colors represent
the 16S rRNA genes from different organisms. We then amplify all the 16S rRNA genes
by PCR.
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Step 2: PCR amplification of community 16S rRNA genes
PCR using universal bacterial primers (8F and 1492R)
Many copies
Imagine we had 4 types of bacteria in our original sample (brown, blue, green and red)
and that the “brown” bacteria were the most abundant. If we assume all else to be equal
in PCR, then after amplification, we should have many copies of the original 16S rRNA
genes and in particular, we should have lots of “brown” bacterial 16S rRNA. All this
amplified product is in our PCR tube and now needs to be separated and analyzed.
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Step 3: Ligation
vector
Amplified 16S rRNA gene
PCR product
ligation
In this step, your amplified products are ligated with a vector. A vector is an linear piece
of DNA, in this case, we are using the TOPO ?? Vector from Invitrogen. After the
ligation reaction, the vector and product for a closed loop of DNA called a plasmid. The
plasmid can then be taken up by specially prepared bacterial cells. Sometimes, the vector
will close without incorporating a piece of DNA. In this case, the plasmid is said to be
without “insert.”
Step 4: Transformation
A.
LB Medium with
ampicillin and X-gal and IPTG
B.
C.
Transformation into competent E. coli cells
The plasmids from the above step are transformed into competent E. coli cells; cells that
have been specially prepared so that they are more willing to take in plasmids. If an E.
coli cell contains a single plasmid with insert, it gains ampicillin resistance and grows as
a white colony (case A). If the cell has a plasmid without insert, it grows as a blue
colony (case B). If the cell does not take up the plasmid, then it does not gain ampicillin
and will not grow until the amplicillin has been degraded and thus comes up much later
on the plate and is called a satellite colony (case C). Without the ampicillin, there would
be no selective pressure for the cells to keep the plasmids.
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TA Cloning:
At the end of your PCR cycle, there is a 10 minute final extension time at 72oC. In this
time, Taq polymerase adds an extra “T” to the PCR products. With time, these extra T’s
drop off. The TOPO TA cloning kit, takes advantage of this property of Taq and has
complementary A’s at the ends of its vector. Thus, the ligation of the PCR product into
the vector is highly favorable since the T and A (hence the name “TA cloning”) will
become paired. If your PCR product is old (over 12 hours), the T’s drop off and the
ligation and thus cloning efficiencies are lowered. For this reason, you will be provided
with fresh amplification reactions from your templates at the start of class.
After cloning, you effectively separate your PCR products spatially on an agar plate
within the individual bacterial cells you spread!
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Pre-Lab Exercise 5:
Refer to the INVITROGEN protocol handout for some of these questions.
1.
Why is the cloning kit called “TA” cloning (1 point)?
2.
What is a vector (1 point)?
3.
What is ligation (1 point)?
4.
Why do we need to work with fresh PCR product (1 point)?
5.
What are the conditions for our ligation reaction (duration and temperature) (2
points)?
6.
What are the conditions for heat shocking our cells (duration and temperature) (2
points)?
7.
What would happen if we forgot to use plates with ampicillin (1 point)?
8.
Why is salt added to the cloning reaction (1 point)?
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Laboratory Exercise :
Supplies (per person unless otherwise stated):
 Bunsen burner
 Ethanol spray bottle
 Paper towels
 Kim wipes
 Flint
 Waste container
 Laboratory tape
 Ethanol resistant marker
 Bucket of ice
 Thin walled 0.2ml PCR tubes
 Pippettes, 0.5-10 l, 5-50l, 20-200l, 100-1000l (1 set per 2 persons)
 Pippette tips for all volumes
 Rack for 0.2 ml thin walled tubes
 Stopwatch
 Heat block at 42 oC (one for the entire class)
 500 ml Erylenmyer flask that will fit onto 37oC shaker table
 37oC shaker table
 37oC incubator
 LB +Amp +IPTG +X-gal plates (3 per student)
 Sterile 5mm glass beads
Solutions:
 TOPO cloning kit with competent cells
 Fresh PCR product with gel photograph
In order for cloning to be successful, you need fresh PCR product. This morning, your
reactions were duplicated so you would have fresh product to clone this afternoon. The
protocols here require GENTLY mixing… DO NOT PIPPETTE UP AND DOWN!!!
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Have your fresh PCR tubes on ice.
Place a 0.2 ml tube rack on the ice with two new 0.2 ml thin walled
tubes.
First you will need to do a ligation of your PCR product and the
vector. Label your tubes with your initials and the contents of your
sample (e.g. TMS-CM-10)
Take TOPO sterile water, salt solution and SOC medium from the
refrigerator racks and place them on ice.
Take one tubes of competent cells from the head of the class and label
them with your initials and experiment information.
Add 2.5 l sterile water from the TOPO TA cloning kit to your empty
0.2 l tube.
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Add 0.5 l sterile salt solution to each tube.
Add 0.5 l of your PCR product to the appropriate tubes.
When ready, come to the head of the class and I will GENTLY add
0.5 l of TOPO vector to your tube (this reagent is VERY expensive).
Once you’ve added the vector, return to your bench, gently flick the
ligation tube to mix the contents and collect them at the bottom of the
tube, place your ligation tubes at room temperature (25oC) and start
your stopwatch.
After 5 minutes at room temperature, return your ligation reaction to
ice.
Gently add 2 l of your ligation reaction to your now thawed
competent cells and gently flick the tubes to mix. DO NOT PIPETTE
UP AND DOWN, YOUR CELLS AND REACTION ARE
SENSITIVE TO THIS!
Place the cells on ice for 5 minutes.
Walk you cells over to the 42oC heat block. Place your cells at 42oC
to “shock” them for EXACTLY 30 seconds. Then return the cells to
ice immediately.
Return your cells to your bench on ice and add 250 l of SOC
medium (also in the refrigerator) to the cells.
Place the tubes of cells in an empty flask and shake at 37oC for 1
hour.
Label 3 LB +Amp plates with your names and sample information.
After the hour is up, plate 30 l, 60l and 90 l of your cells onto the
plates. Use your sterile glass beads to spread the cells as we learned
in lab #1 with the sterile water.
Place the plates at 37oC overnight.
We will revisit these plates tomorrow.
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Report Guidelines for Laboratory 5:
1. What PCR product did you clone (5 points)?
2. How long did you incubate at room temperature, what would you expect if
you incubated longer (8 point)?
3. Why did you plate 3 different volumes (2 points)?
4. Briefly outline the steps in the above protocol (10 points).
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