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Lab 3 Lab Manual

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Lab 3 Lab Manual

Uploaded by

makabigail7
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© © All Rights Reserved
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BIOL2313 Genetics Laboratory 2023-2024

Exercise 3
Transformation of Escherichia coli

Bacterial transformation is a technique to introduce a foreign plasmid (usually carrying our


gene of interest) into bacteria in order to get large quantities of it. In the calcium chloride/heat
shock transformation method, the plasmid DNA is first mixed with competent cells (cells that
are ready to take up DNA) in calcium chloride solution and incubated on ice for some time.
Then the mixture is subjected to a brief heat shock, which helps the uptake of DNA. The cells
are then recovered and spread onto suitable antibiotic-containing media. The antibiotic added
provides a selective pressure because only the bacteria that have acquired the plasmid
containing the same antibiotic-resistant gene can survive.

In this exercise, you are going to transform Escherichia coli with two plasmids assigned to
your group. Each plasmid carries either the ampicillin- or the kanamycin-resistant gene as the
selectable marker. At the end of the experiment, report which antibiotic resistant gene the
respective plasmid carries.

Learning outcomes:

After this lab session, you should be able to:


1. Understand the principle of calcium chloride and heat-shock transformation.
2. Understand the basis of antibiotic selection.
3. Be aware of the applications of bacterial transformation.
4. Transform E. coli with a given plasmid.
5. Familiar with the aseptic and spreading plate techniques.

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BIOL2313 Genetics Laboratory 2023-2024

Procedures:
1. Thaw and keep 3 tubes of competent cells (25 µl each) on ice. Label these tubes with
“A” (i.e. with Plasmid A), “B” (i.e. with Plasmid B) and “–” (i.e. without plasmid
DNA), respectively.
2. Prepare a Bunsen flame.
3. Using aseptic techniques, add 5 µl of the plasmid solution (1 ng/µl) labeled “A” and
“B”, to the labelled “A” and “B” tubes that contain competent cells, respectively.
4. Place the tubes on ice for 30 min.
_____________________________________________________________________
5. Heat shock the cells at 42°C for 45 sec.
6. Place the tubes on ice for 2 min.
7. Prepare a Bunsen flame.
8. Using aseptic techniques, add 475 µl of LB to each tube of the heat-shocked
competent cell.
9. Incubate the tubes at 37°C with shaking (500 rpm) for 45 min. Label the plates
according to the following table:
Plate # 1 2 3 4 5 6 7
Agar plate LB LB/amp LB/kan LB/amp LB/kan LB/amp LB/kan
Plasmid DNA - - - A A B B
(A, B or -)

_____________________________________________________________________
10. Spread 100 µl of cells onto respective plates.
11. Incubate the plates at 37°C overnight.
12. Observe the growth of the cells on the different plates. Record your data.

Calculation of bacterial transformation efficiency

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BIOL2313 Genetics Laboratory 2023-2024

To know more about the calcium chloride/heat shock transformation method, see the
animation at

http://www.dnalc.org/ddnalc/resources/transformation2.html

Reference figures:

Figure 1. The illustration of bacterial transformation with antibiotic resistance gene.

Lab 3 activities:

Prelab talks – 30 minutes

Preparation of cells and plasmids – 15 min

Briefing on heat shock transformation – 15 min

Heat shock of cells – 15 min

Wait for recovery and briefing on aseptic techniques – 45 min

Spread the plate with antiseptic techniques – 60 min

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