GENE TRANSFER IN E. COLI AP LAB

TRANSFORMATION PROTOCOL

  1. Label one of the plastic tubes + (with plasmid) and the other plastic tube, - (without plasmid).
  2. Add ice to each of the Styrofoam cups. Place a labeled tube in each cup.
  3. Use a sterile pipet to add 250 μL of ice-cold CaCl2 to each tube. Please Note: Pressing the conical area between the stem and bulb of the pipet provides better control of the amount of liquid being aspirated. Place used pipet in the beaker filled with bleach solution.
  4. Flame the metal loop. When cool, use the loop to pick up E. coli cells from the plate provided. Skim the loop across the surface of the agar and pick up a line of bacteria about 2X the diameter of the loop. Be careful not to pick up any agar with the loop.
  5. Immerse the cells on the loop into the CaCl2 in the + plasmid tube and vigorously spin the loop in the solution to dislodge the cells.
  6. Immediately pulsate the cells with a sterile pipet. It is not necessary to draw the suspension up into the tube; keep the solution in the stem of the pipet. Examine the tube to confirm no visible clumps of cells remain in the tube. The suspension should appear milky white. Place the used pipet in the beaker filled with bleach solution.
  7. Return the tube to ice.
  8. Repeat steps 4-7 for the – plasmid tube.
  9. Use aplastic sterile loop to obtain 10 μL(1 loopful) of the assigned plasmid to the tube labeled +. The DNA solution should form a film across the loop opening. Immerse the loopful of plasmid directly into the cell suspension and spin the loop to mix the DNA with the cells. Place loop in beaker filled with bleach solution.
  10. Incubate both tubes on ice for 15 minutes.
  11. While the tubes are incubating, label the six plates provided with your period number – table number. Label one LB plate, one LB/Amp plate, and one LB/Kan plate with a +(with a plasmid) and one LB plate, one LB/Amp plate, and one LB/Kan plate with a – (without a plasmid).
  12. At the end of the 15 minute incubation time, heat shock the cells as follows:
  13. Take tubes from ice to hot water bath on side counter.
  14. Use the thermometer to make sure the hot water bath is at 42ºC. Make adjustments using ice, etc.
  15. Place both tubes in the water bath and immerse them for 90 seconds.
  16. Gently agitate the tubes while they are in the water bath.
  17. At the end of 90 seconds, immediately place tubes back on ice for at least one minute.
  18. Use a sterile transfer pipet to add 250 μL of Luria broth to each tube, taking care not to touch the sides of the tube or the solution inside. Gently tap the tubes with your finger to mix the LB with the cell suspension. Place pipet in bleach solution.
  19. Allow the tubes to sit at room temperature for an 8 minute recovery.
  20. Inoculate the plates as follows, using the + tube for all plates labeled +, and the – tube for the plates labeled -.
  21. Place the plates, agar-side down on the lab table.
  22. Use a sterile pipet to add 100 μL of cells from the plastic tube to each of the appropriate plates.
  23. Carefully pour 4-6 glass beads onto the agar of each plate where you added the 100 μL of cells.
  24. Spread the bacteria evenly across the plate by using a back-and-forth motion (not swirling round and round) to move the glass beads across the entire surface of each plate. Do this for several minutes.
  25. Let plates sit for several minutes to allow agar to absorb the cell suspension.
  26. Remove glass beads by holding plate vertically over the bleach beaker and allowing the beads to fall into the bleach. Be careful not to splash the bleach.
  27. Place all plates in the incubator, agar-side up.
  28. Clean-up:
  29. Discard vials of Luria Broth and CaCl2in the trash.
  30. Use masking tape to seal plastic tubes. Place in bleach bin on side counter.
  31. 6th Period Only → Tape E. coli plate and place in bleach bin.
  32. Throw away all paper trash.
  33. Discard ice but do not throw away Styrofoam cups!
  34. Wipe down table top with 409.
  35. Wash hands thoroughly.