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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.

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procedure
Procedure
  • The Plasmid pBestluc

and the CaCl must be kept on ice

throughout the entire experiment.

slide3
Obtain the plate of Luria agar with the

actively growing E. coli lawn on it.

  • Obtain a microcentrifuge tube and using a sterile 1m pipette, add 0.25ml (250ul)

of ice cold CaCl to the tube

slide4
3. Using a sterile inoculating loop, remove a loopful of E. coli from the surface of the agar. (DO NOT REMOVE ANY AGAR!!!)
  • 4. Place the loop of collected E. coli into the CaCl and twirl rapidly
  • 5. DISPOSE OF THE LOOP IN A BIOHAZARD BAG
slide5
6.Add 10ul (10 microliters) of the plasmid (pBestluc) using a yellow-tipped plunger micropopette, to your microcentrifuge tube.
  • Cap tube and gently tap with your finger to mix.
  • Incubate your tube on ice for 15 minutes.
slide6
9. While the tube is on ice, obtain your LB + ampicillin plate.

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

slide7
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.

slide8
14. Remove tubes from bath and place on ice for 2 minutes
  • 15. Remove tubes from ice and add 0.25 (250 ul) of room temeprature Luria Broth using a sterile pipette.
  • 16. Finger-tap to mix.
  • 17. Tube may now be kept at room temp.
slide9
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)

slide10
Gently rock the plate in order to evenly spread the solution.
  • Let sit undisturbed for 1 hour. Place in incubator,

inverted - overnight at 37oC.

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