(Students follow the procedure below using
Student Study Sheets)
DAY 1:
WEAR SAFETY GOGGLES AND EITHER A LAB COAT OR APRON FOR ALL THESE STEPS:
(1) Your teacher will provide you with a capped test tube
containing a rapidly-growing culture of E. coli in a
liquid nutrient broth.
(2) You will also receive four petri plates: two containing
plain nutrient agar (marked "P") and two with
nutrient agar containing tetracycline (marked "T").
(3) Using sterile techniques explained by your teacher,
streak one "P" and one "T" plate by immersing the
sterile swab (only) into the broth medium in the
culture tube, then streaking as your teacher directs.
Mark each of these plates with a (*).
(4) Store the streaked plates upside down in an incubator
or oven at 37oC.
(5) Pour the remaining E. coli in liquid broth from the
capped culture tube into a 15 mL centrifuge tube.
(6) Follow your teacher's directions and centrifuge the
tube for five minutes.
(7) Decant the supernatant (the clear fluid on top of the
"button" of cells). Do that by pouring the supernatant
carefully into a beaker containing 10% Chloroxreg.
solution (1 mL household bleach to 9 mL water). Your
teacher may ask you to use a pipet to remove the
supernatant.
(8) Use a 5 mL pipet to add 2 mL of CaCl2 to the button of
cells at the bottom of the centrifuge tube.
(9) Mix the bacteria and CaCl2 solution well, but gently.
(10) Add another 6 mL of the CaCl2 solution, in 2 mL
increments, to the centrifuge tube with the same pipet
used in Step 8.
(11) Place the test tubes in an ice bath for 30 minutes.
(12) At the end of 30 minutes, centrifuge the tube again for
five minutes.
(13) Decant the supernatant using the same procedure you
used in Step 7.
(14) Use a 1 mL pipet and add 0.75 mL of cold CaCl2 to the
button of cells at the bottom of the centrifuge tube.
(15) Mix the cells and the CaCl2 gently and store in the
refrigerator overnight.
(16) Wash your hands thoroughly before leaving the lab.
DAY 2
WEAR SAFETY GOGGLES AND EITHER A LAB COAT OR APRON FOR ALL THESE STEPS:
(1) Use a 1 mL pipet to divide the liquid in the centrifuge
tube evenly into two small, capped tubes called
microfuge tubes. For the rest of this procedure you will
work in groups of two.
(2) Take your microfuge tube to your teacher so he or she
can add two drops of a solution containing plasmids to
each of your tubes.
(3) Cap each microfuge tube and gently mix the contents
by inverting the tube two or three times.
(4) Place the tubes in an ice bath for 30 minutes.
(5) At the end of 30 minutes, place the tubes in a 37oC
water bath for five minutes.
(6) At the end of five minutes, use a sterile 5 mL pipet to
add 1 mL of sterile nutrient broth to the microfuge
tube. This longer pipet is used to reduce the chance of
contamination.
(7) Gently mix the contents of the tube and place in an
incubator or oven at 37oC for 45 to 60 minutes. Mix the
tube contents frequently during this period.
(8) Using a sterile 1 mL pipet, place 0.1 mL of the mixture
in the microfuge tube on a tetracycline agar plate and
0.1 mL mixture on a plain nutrient agar plate. Mark
each of these plates (**).
(9) Incubate the plates (inverted) for 24 to 48 hours and
observe results.
(10) Dispose of all contaminated materials as directed by
your teacher.
(11) Wash your hands thoroughly before leaving the lab.
AE Partners Collection Index
