Bacterial transformation protocol

Bacterial Transformation

This is a protocol outline for transformation of bacteria (i.e. inserting a DNA plasmid into the bacterial cell). Protocol

Materials

  • 2, 15mL snap-cap media tubes
  • CaCl2 solution
  • pGFP plasmid (0.005μg/μl)
  • LB and LB/amp petri dishes
  • MM294 E.coli bacterial culture (on LB petri dish)
  • Water bath (42 oC)
  • LB liquid medium
  • Ice
  • Micropipettes/tips
  • Sterile loops
  • Sterile glass beads

Steps

Note

Until step 10 all reagents should be kept as cold as possible on ice.

  1. Label sterile 15mL tubes with your initials, and label one tube "+" and the other "-".

  2. Add 250μl of CaCl2 to each tube; place both tubes on ice. Until step X, keep tubes on ice as much as possible

  3. Using a sterile loop, transfer a clump (2-3 mm in diameter) of bacteria to the "+" and "-" tube. Avoid cross-contaminating tube contents.

  4. Use a pipette to breakup the bacterial clump and disperse in the CaCl2 solution in each of the 15mL tubes.

  5. To the "+" tube only, add 10μl of 0.005 μg/μl pGFP plasmid directly into the liquid. Tap the tube using your finger to mix.

  6. Incubate 15mL tubes on ice for ~ 15 minutes.

  7. During step 6 incubation, label 4 petri dishes (be sure to write on the edge of the plate):

    Petri dish Label
    LB "+"
    LB "-"
    LB/amp "+"
    LB/amp "-"

  8. Add your initials to each plate and the date.

  9. After the ice incubation, keep the tubes on ice and bring them to the 42 oC water bath. Place the tubes into the warm water for 90 seconds, then immediately return the tube to the ice.

  10. Add 250μl of LB medium to each tube, being careful not to cross-contaminate.

  11. Add 100μl of the "+" and "-" tube contents to the respective labeled petri dishes. Immediately after adding 100μl to the center of the agar plate, use sterile beads to spread the bacteria.

  12. Store plates upside down at 37 oC overnight.

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