Bacterial Transformation

Overveiw:

The goal of this Lab will be to make a plasmid that confers ampicillin antibiotic resistance to E. Coli.
That plasmid that we will use is pUC18 and it comes naturally in E.coli. Transformed bacterial cells
will grow while bacteria cell will not.

Objective:

1. Transform bacteria pUC18 in E.coli. resistant to ampicillin.

2. Identify transformed bacterial cells
3. Calculate transformation efficiency of bacteria

Preparation and Procedure:

Before the Experiment (about 48 hours)

1. Divide petri plates into one group of 17 to be poured with Luria broth agar, adn another group
of 16 to be poured with Luria broth agar containing ampicilin. Clearly mark them
2. Open the cap on the Luria broth agar bottles and place them in microwave. Heat teh agar in 1
minute intervals until it is completely liquefied.
3. Remove
4. Open a petri plate lid as little as possible and pour enough agar approximately into the plate to
cover the bottom. close the lid. Repeat this with the 16 other dishes.
5. When the second Luria broth bottle has settled and cooled but not set, pour in the ampicillin
powder provided with the kit. gently invert the bottle a few times to mix the ampicillin with the
agar--DONT SHAKE!!!
6. Alow the plates to harden undistributed. Refrigerate until needed.

The day of the experiment.

1. Obtain 2 poured Luria broth agar plates. Label one of the plates "LB+" and the other "LB-"
2. Obtain Obtain 2 poured Luria broth agar with ampicillin plates. Label one of the plates
"LB/amp+" and the other "LB/amp-"
3. Obtain two 2mL microfuge tubes. Label one of the tubes + and the other -
4. Using a 1mL micropipet , add 250 ul of ice cold 0.05M CaCl2 to each of the sterile microfuge
tubes.
5. Using a sterile, plastic inoculating loop, add 2-3 E.coli colonies to each of the two 2mL
microfuge tubes. Try not to gouge the agar that the I.coli are growing on as no agar shoudl be
introduced to the tubes. tap teh inoculating loop firmly against eh side.
 6. Use the micropipet from step 4 to suspend the cells in the CaCl2 in the tubes. Do this by
alternately drawing the solution from the tube into the micropipet and emptying it several times.
7. Using a digital or fixed micropipet, introduce the antibiotic-resistance plasmid by adding 10ul
of pUC18 solution into the + tube, and mix the content by tapping on the tube.
8. Place both tubes in ice container for 15 minutes
9. heat shock the bacterial cells by placing both tubes in a 42 degree C for 90 seconds
10. Return both tubes immediately to the ice container, and allow them to remain there for 2
minutes.
11. Using one sterile, plastic 1mL micropipet add 250 ul of Laria broth to each of the microfuge
tubes and tap on each tube to mix the contents. Place the tubes in the microfuge tube rack at
room tem
12. p.
13. Using a digital or fixed volume micropipet, draw 100 ul of + cells and dispense them onto the
"LB+" plate. Place 100 ul of "+" cells on the "LB/amp+" plate also.
14. Use a sterile spreading rod to spread the "+" plates. Cover the plates immediately and place
them aside to set for a few minutes. Resterilize the spreading rod with alcohol and flame.
15. Using a clean tip on the digital or fixed volume micropipet, draw 100 ul of the "-" cells and put
them on the "LB-" plate and the "LB/amp-" plate.
16. Spread
17. Tape all plates shut using clear tape. incubate the plates upside down overnight in 37degree C
incubator.

Results:

Conclusion: