Transformation with Firefly gene Lab Protocol. Procedure. The Plasmid pBestluc and the CaCl must be kept on ice throughout the entire experiment. Obtain the plate of Luria agar with the actively growing E. coli lawn on it.
and the CaCl must be kept on ice
throughout the entire experiment.
actively growing E. coli lawn on it.
of ice cold CaCl to the tube
6.Add 10ul (10 microliters) of the plasmid (pBestluc) using a yellow-tipped plunger micropopette, to your microcentrifuge tube.
10. Using forceps or wearing gloves place a sheet of nitrocellulose paper on the surface of the agar.
11. Lift the lid only enough to place the Nitrocellulose paper on the agar and replace the lid ASAP
12. The bacterial cells must be HEAT SHOCKED in order to allow for plasmid transformation to occur.
13. After 15 min on ice, remove the tube and immediately place in a 42oC water bath for 75-90 seconds.
14. Remove tubes from bath and place on ice for 2 minutes allow for plasmid transformation to occur.
18. Using a clean pipette, add 20ml (200ul) of your plasmid/bacteria solution to the center of your Luria Agar plate covered with nitrocellulose paper. Cover the petri dish
** Repeat step 18 with your LB Agar Plate (w/o Ampicillin). DO NOT USE NITROCELLULOSE PAPER FOR THIS DISH)
inverted - overnight at 37oC.